Calibration of HPLC:

High-Performance Liquid Chromatography Calibration Procedure

HPLC Calibration frequency:

  • Calibrations shall be performed Six monthly.

Calibration of High-Performance Liquid Chromatography (HPLC) consists of the following parameter,

  1. Calibration of pump by flow-rate accuracy measurement.
  2. Calibration of pump by gradient flow measurement.
  3. Calibration of column oven and sample cooler for temperature accuracy.
  4. Calibration of UV-VIS / PDA detector by reference energy measurement by lamp.
  5. Pressure Test.
  6. Calibration of Drift and Noise.
  7. Calibration of detector for Linearity of response.
  8. Calibration of auto sampler for carry over check.
  9. Calibration of auto sampler for linearity of injection volume.
  10. Calibration of detector for wavelength accuracy.
Calibration of HPLC
Calibration of HPLC

HPLC Calibration Procedure:

  • Calibration parameter shall be performed as described below,

1. Calibration of pump by flow-rate accuracy measurement

  • Mobile Phase: HPLC grade water or equivalent (Which is filtered with 0.45 μ filter and degassed).
  • Flow rate: 0.5 mL/min, 1.0mL/min and 3.0mL/min.
  • Connect the restricted capillary in place of column connections with the instrument.
  • Set the vacuum degassor (if installed) to run continuously, or set the purge rate to 100 %.
  • Keep HPLC mobile phase in the respective reservoir.
  • Purge the system for each flow line with filtered HPLC grade water and allow running about 15 min. prior to check the flow.         
  • Ensure that the tubing from the reservoir to column inlet shall be free from air bubble and system pressure should be constant.  
  • Take 10 mL clean and dried volumetric flask and keep the restricted capillary’s outlet in such a way that when the pump is on the HPLC grade water drops, falls into volumetric flask.
  • Care should be taken that the water drops should not touch the walls of the flask.
  • Set the pump for 0.5 mL/min. flow-rate, start the pump and allow running about 10 min. Then keep the flask at restricted capillary’s out let, start the pump and then simultaneously start stop watch.
  • Stop the watch when volume of HPLC grade water reached at the mark in the volumetric flask. Derive the time taken and make entry in the format.
  • Repeat the above procedure for flow rate of 1.0 mL/min and 3.0 mL/min in 10 mL volumetric flask and record.     
  • Verify the results against the acceptance criteria and in case of any non-compliance, inform to Department head or designee.
  • Acceptance Criteria: Actual time observed should be within ±2.0% of the theoretical time.

Related: Principle of HPLC (Liquid Chromatography), Difference Between C18 and C8 HPLC Column

2. Calibration of pump by gradient flow measurement.

  • Mobile phase preparation:
    • 1. Mobile phase A (A=B if pump is quaternary) Take about 1000 mL of HPLC grade water filtered (with 0.22 µ filter) in cleaned and dried mobile phase bottle and sonicate it for about 10min.
    • 2.Mobile phase B (C=D if pump is quaternary): Take 5.0 mL of Acetone in 1000 mL volumetric flask, dilute to 1000 mL with HPLC grade water. Filter it with 0.22 µ filter paper and degas by sonication for about 10 min.
  • Chromatographic conditions:
Column:Restricted capillary
Flow Rate:2.0 mL/min.
Wavelength:265 nm
Injection volume:10 µL
Run time:30 minutes (20 min if pump is quaternary)
Sample cooler temperature:NA
Column ovenNA or Set as 25°C
  • Sample/standard preparation: Take HPLC grade water or equivalent as a standard/sample.      
  • If a column is attached to the instrument, remove it and connect restricted capillary.
  • Purge the system with HPLC grade water, which is previously filtered through 0.45µ filter.         
  • Take the HPLC grade water previously filtered through 0.45 µ filter and use as mobile phase for Pump-A (Use same mobile phase for pump A and pump B at the time of pump calibration using quaternary gradient). 
  • Prepare 0.5 % v/v solution of Acetone in HPLC grade water and filter through 0.45 µ filter and use as mobile phase for the Pump – B (Use same mobile phase for pump C and pump D at the time of pump calibration using quaternary gradient).          
  • Set the system gradient flow program as per below table for quaternary Waters make HPLC system.
Time (in min)Flow%A%B%C%DCurve
Initial2.0 mL505000
22.0 mL00505011
62.0 mL50500011
102.0 mL454510011
122.0 mL50500011
142.0 mL454501011
162.0 mL50500011
202.0 mL50500011
  • Inject HPLC grade water or equivalent as a sample for five (5) times and record peak height / area.           
  • Plot a linearity curve of peak height% (theoretical) v/s observed peak height (µv) using least square method. Calculate the correlation coefficient (r) and record the observations.         
  • Calculation:
    • 1.   (Mean of second peak height  X 100) /Mean of first peak height      = 
    • 2.   (Mean of third peak height X 100)/Mean of first peak height =       
  • Acceptance Criteria : Calculation should be between 9.5% and 10.5%. RSD of 5 injection should be NMT 2.0%.    

3. Calibration of HPLC Column Oven and Sample Cooler for temperature accuracy.

  • Carryout the calibration for the temperature point at 30.0°C and 60.0°C and 80.0°C (depends upon the capacity of column oven with respect to system configurations) using digital temperature indicator.            
  • Set the temperature of column oven at 30°C, keep the temperature probe in the centre of column oven and allow to stabilize the set temperature for at least 15 to 20 min. 
  • If column oven is available with cooler, Set the temperature of column oven at 15°C, keep the temperature probe in the centre of column oven and allow to stabilize the set temperature for at least 15 to 20 min.  
  • Check the displayed temperature of column oven and temperature indicator device.
  • Also carryout for 60°C and 80°C temperature calibration (depends upon the capacity of column oven with respect to system configurations) in the same manner and note all the reading in observation table.  
  • Set the sample cooler temperature for 4°C and 25°C and repeat the same procedure.

            Note: Thermometer used should be calibrated to at least ± 2 °C or better.

  • Acceptance Criteria: ± 2°C of set temperature.

4. Calibration of UV-VIS/PDA detector by Reference energy measurement of reference energy measurement by the lamp.

  • This is only applicable to waters make HPLC as on detector part user can see the intensity of the UV-VIS/PDA light.
  • Check the Reference Energy for Lamp in PDA detector/UV-Visible lamp at 225nm.
  • Acceptance Criteria:
    • UV-Visible: NLT 15 nA
    • PDA (W2996) NLT 10000 nA
    • PDA (W2998) NLT 10000 counts

5. Calibration of HPLC by Pressure Test (HPLC calibration waters)

  • Pressure test to be carried out before pump calibration.
  • Purge the system for each flow line with mobile phase and use any of the flow line for the pressure test.
  • Ensure that the tubing from the reservoir to the column inlet shall be removed.
  • Plug the outlet of the pump using a dead nut.
  • Set the flow rate of the pump to 0.1 mL/min.
  • Observe the pressure until it reaches the maximum psi/bar. When the pressure reaches to the maximum limit, then leakage in the pumping system is observed which implies good check valve performance.
  • Repeat the same procedure for further two more times and observe the same.
  • In case the pressure do not reach the maximum and fluctuates at some psi/bar, implies poor check valve performance or leaks within the pumping system.
  • After satisfactory completion of the pressure test, proceed for further calibration parameters.
  • Verify the results against the acceptance criteria and in case of any non-compliance, inform to department head or designee.
  • Acceptance Criteria: Maximum Pressure Limit should be as per company qualification.

6. Calibration of Drift and Noise

  • Mobile phase preparation: Mix 700 mL of HPLC grade water and 300 mL of Methanol and filter through 0.22 µ nylon filter, degas for 10 min. by sonication.           
  • Chromatographic conditions:
Column:BDS HYPERSIL C18 (25 cm x 4.6 mm, 5 µ) or equivalent.
Flow Rate:1.0 mL/min.
Wavelength:254 nm (For UV) 254 nm (2D data collection at 3.6 nm bandwidth)- (For PDA)
Injection volume:Inject immediate standard
Run time:15 min.
Sample cooler temperature:NA
Column ovenNA or set as 25°C
  • Flush the system with water for 15 minutes. 
  • Ensure that, the column is conditioned before injecting the sample.
  • After conditioning the column equilibrate the system for 15 min and then run three immediate standard verification as per test condition for 15 min.
  • Observe system equilibration and pressure graph of immediate standard verifications.
  • If immediate standard verifications are found satisfactory then run immediate standard for obtaining drift and noise as per test condition for 15 min. In case of not satisfactory observation, consult service engineer.         
  • After “inject immediate standard” injection run completion, process the data using processing method as the average peak to peak noise is calculated based on a 30 second noise interval from 3 to 15 minutes and is reported in mAU.
  • The detector drift is calculated based on a 30 second noise interval from 3 to 15 minutes and is reported in mAU/Hr.
  • Record the results in observation.
  • Acceptance Criteria:
DetectorNoise LimitDrift Limit
For UV DetectorLess than or equal to 60 µAULess than 10 mAU/ Hr
For PDA DetectorLess than or equal to 80 µAULess than 10 mAU/ Hr

7. Calibration of Detector for Linearity of Response

  • Mobile Phase Water and Acetonitrile (85: 15): Take 850 mL HPLC grade water and 150 mL HPLC grade Acetonitrile, filter it through 0.22 µ filter paper and sonicate it for about 10 min.     
  • Chromatographic conditions:
Column:BDS HYPERSIL C18 (25 cm x 4.6 mm, 5 µ) or equivalent.
Flow Rate:1.0 mL/min.
Wavelength:272 nm
Injection volume:20 µL
Run time:10.0 min.
Sample cooler temperature:NA
Column ovenNA or set as 25°C
  • Prepare five concentrations solution of caffeine as 20 ppm, 40 ppm, 60 ppm, 80 ppm and 100 ppm.                       
  • Set up the instrument as per the conditions.  
  • Ensure that, the column is conditioned before injecting the sample.
  • Inject the all five samples in duplicate and record the area of principal peak in observation table.
  • To verify proper system saturation, three injection of same concentration to be injected, and if the area is precise, then only proceed for further injections of detector linearity.      
  • Plot a linearity curve of concentrations Vs corresponding mean area, using least square method. Calculate the correlation coefficient (r), and record the observations .     
  • Acceptance Criteria:
    • Correlation co efficient (r)= NLT 0.999,
    • Intercept: NMT 3.0% of 60 ppm area

8. Calibration of Auto Sampler for Carry Over Check

  • Mobile Phase preparation:
    • 1. Mobile phase A: Take HPLC grade water 1000 mL in 1-liter bottle filtered with 0.22 micron filter in cleaned and dried mobile phase bottle.
    • 2. Mobile phase B : Take HPLC grade Methanol 1000 mL in 1-liter bottle. filtered with 0.22 micron filter in cleaned and dried mobile phase bottle.
  • System requirements:
Column:X bridge C18 (50 x 4.6 mm), 3.5µ or equivalent
Flow Rate:1.0 mL/min.
Wavelength:273 nm
Injection volume:10 µL
Run time:2.0 min. (For preblank and sample preparation-2 (Concentration 4 ppm) and post blank injection.
Run time:15.0 min. (For sample preparation-1 (Concentration 4000ppm)
Sample cooler temperature:NA
Column oven35°C ± 5°C
Sampling rate:5 points /Sec
Time constant:0.4 (Sec)
Needle WashWater: Methanol (70:30)
  • Gradient flow programming table as shown in below,
Time (min.)Flow (mL/min.)A/B/C/DA/B/C/DCurve*
Initial1.070306
2.501.070306
3.001.001006
7.001.001006
7.501.070306
15.001.070306

*Mainly shown in Waters makes HPLC.

  • Preparation of Diluent (Solution A): Take HPLC grade Water 700 mL filtered with (0.45micron filter) and take HPLC grade Methanol 300 mL filtered with (0.45 micron filter) and mix well and degas it for 10 min.
  • Sample preparation-1 (Conc. 4000 ppm): Weigh accurately 40 mg of Caffeine and transfer it in 10 mL clean and dried volumetric flask, dissolve in and dilute up to the mark with diluent (Solution A).           
  • Sample preparation-2 (Conc. 4 ppm): Taken 4 mL of above solution A and transfer it in 200 mL clean and dried volumetric flask, dissolve in and dilute up to the mark with diluent. Taken 5 mL of above solution and transfer it in 100 mL clean and dried volumetric flask, dissolve in and dilute up to the mark with diluent.
  • Setup the HPLC System with the above conditions and keep the flow for 30 minutes to saturate the column with the mobile phase.          
  • Inject two injections of 10.0 µL diluent as pre-blank.           
  • After getting the baseline from the blank injection, inject three (3) injection of 4 ppm of caffeine solution (Sample Preparation -2) and record the chromatogram and calculate mean area.
  • Inject one injection of 4000 ppm of caffeine (Sample Preparation -1) and record the chromatogram.
  • Inject one injection of 10 µL diluent as post-blank, if any peak observed at the RT of principal peak, calculate the carry over by following formula.

Calculation:

%Carryover =  (Area of carry over Caffeine in post blank X 0.01) ÷Mean area of Caffeine peak in Sample Pre. -2

  • Acceptance Criteria: % Carry over should not more than 0.01%   

9. Calibration of Auto Sampler for Linearity of Injection Volume

  • Mobile phase preparation: HPLC grade Water : Acetonitrile ( 85:15) : Mix  850 mL of HPLC Water and 150 mL of Acetonitrile and filter through 0.22 µ nylon filter. Degas it for 10 min. by sonicator.           
  • System requirements:
Column:BDS HYPERSIL C18 (25 cm x 4.6 mm, 5 µ) or equivalent
Flow Rate:1.0 mL/min.
Wavelength:272 nm
Injection volume:10 µL
Run time:10 min. (Retention time: About 6.0 min)
Sample cooler temperature:NA
Column ovenNA
  • Sample/Standard preparation:
    • Sample preparation (Stock solution): Weighed accurately 60 mg of caffeine and transferred it to 200 mL clean and dried volumetric flask, dissolved in and dilute up to the mark with water.
  • Up to 100µL loop size: Further diluted 5.0 mL of stock solution to 100 mL with water and mixed well.
  • Above 100µL loop size: Further diluted 2.0 mL of stock solution to 200 mL with water and mixed well.   
  • Setup the HPLC System as per above chromatographic conditions and saturate the column with the mobile phase for about 30 minutes.   
  • Fill 5 vials with Sample solutions respectively and place into sample tray. 
  • Vial positions : Place the Vial at  1, 29, 58, 87, 116 position for Waters Alliance system.
Loop CapacityInjection Volume
50µL5µL10µL20µL30µL50µL
100µL5µL20µL50µL80µL100µL
200µL5µL20µL50µL100µL200µL
500µL5µL50µL100µL200µL500µL
  • Place five different vial for replicate injection of 200µl and 500µl injection volume for respective loop size i.e. single injection from each vial.
  • Note: first inject blank and five injections of 5µL and check %RSD it should be within acceptance limit. If it is found satisfactory than start the remaining sequence. In case of non-satisfactory inform to section head or designee.       
  • Record the area due to principal peak. Take the mean area and calculate the %RSD for five replicate results and record the results in the observation .
  • To verify proper system saturation, three (3) injection of same concentration to be injected, and if the area is precise, then only proceed for further injections of auto sampler linearity.
  • Plot a linearity curve of concentrations Vs corresponding mean area, using least square method. Calculate the correlation coefficient (r) and record the observation ..
  • Acceptance Criteria:
    • % RSD should be NMT 2.0% and Correlation coefficient should be NLT 0.999

Related: What are theoretical plate numbers?

10. Calibration of Detector by Wave length Accuracy Measurement

  • Mobile phase preparation:
    • Mobile phase A: Take HPLC grade water in 1-liter bottle. filtered (with 0.22-micron filter) in cleaned and dried mobile phase bottle.
    • Mobile phase B: Take HPLC grade Methanol in 1-liter bottle. filtered (with 0.22-micron filter) in cleaned and dried mobile phase bottle.
  • Diluent:  Take HPLC grade water 700 mL filtered with (0.45micron filter) and take HPLC grade methanol 300 mL filtered with (0.45 micron filter) and mix well and degas it for 10 min. 
  • System requirements:
Column:X bridge C18 (50 x 4.6 mm), 3.5µ or equivalent
Flow Rate:1.0 mL/min.
Wavelength:202 nm to 208 and 270 nm to 276 nm.
Injection volume:10 µL
Run time:2.0 min.
Sample cooler temperature:NA
Column oven35°C ± 5°C
Sampling rate:5 points /Sec
Time constant:0.4 (Sec)
Mobile phase program:70 mobile phase A: 30 mobile phase B
  • Sample preparation:
    • 1. Stock Solution: Weigh accurately about 100mg of caffeine and transfer to 100 mL of clean and dried volumetric flask. Dissolve it and dilute to 100 mL with diluent.
    • 2. 60 ppm Sample: Transfer 6 mL of stock solution to 100 mL clean and dried volumetric flask. Dissolve in dilute to 100 mL with diluent.
  • Set up the instrument as per the given conditions.

Note: Before calibration clean the system with Isopropyl alcohol (IPA).

  • Ensure that the column is conditioned before injecting the sample.
  • Run the system with 202 nm to 208 nm and 270 nm to 276 nm by the increment of 1-nm wavelength and record the chromatograms. i.e. prepare the instrument methods with different wavelengths if the detector is UV.

Note:

  • (A) In the case of PDA detector select the range 195 nm to 280 nm and carry out only one sample single injection in 3D mode. Extract the data of 202 nm to 208 nm and 270 nm to 276 nm by the increment of 1 nm wavelength.
  • (B) In the case of UV detector apply individual wavelength for one injection (if facility available in the system).
  • Record the observed.
  • Find the highest area from the results and consider its respective wavelength as the actual wavelength.           
  • Acceptance Criteria:
    • For 202 nm to 208 nm Range (Maximum area should be at 205 ± 2 nm)
    • For 270 nm to 276 nm Range (Maximum area should be at 273 ± 2 nm)

Care to be taken during the HPLC Calibration:

  • All calibration standards used for the calibration should be procured from authentic sources with certification and it should be either NIST traceable or pharmacopoeial reference standards (wherever possible) or from the authentic sources.
  • If the method parameter given for respective calibration study is not possible to apply in particular HPLC due to different make and different configuration then the alternative method parameter can be used.
  • Weight of Caffeine given in the SOP can be modified as per requirement but the final concentration cannot be modified.

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