hplc calibration method

HPLC is one of the most sophisticated instruments in the quality control department, this machine is used for very précised analytical results, This machine is capable to provide that. Since it is a very sensitive and sophisticated machine, therefore, it requires a good handling and regular calibration practice. In this article, you will find the very practical and easy to perform HPLC calibration method in step to step procedure guidance.

General Cleaning:

Ensure that Power to the Instrument is switched off. Clean daily the outer surface of the instrument with a clean dry cloth and tissue paper, occasionally with a wet cloth dipped in dilute detergent solution may be used. Remove the moisture if any with tissue paper and dry cloth.

Operation of HPLC:

There shall be a plan available for analysis before start up on the instrument

Mobile phase Preparation: The mobile phase shall be prepared as per composition and pH describe in the standard test procedure. The mobile phase container shall have labeling details as follows:-

Product name:      _________

Analysis :             ___________

Composition         ___________

 Prepared by:         ___________

 Used before:         ___________

Details like weight of the buffer observed pH input in balance and pH meter for the mobile     phase preparation shall be recorded.                                 

The mobile phase shall be filtered and visually checked for clarity before use.

Switch on the instrument from mains & connect the instrument to the PC With a USB power cable.

Switch on the instrument, and turn on the PC also, double click on LC SOLUTION icon appears on the window.

The operation window will appear, double click on 1 icon. The administration window will appear click on the OK button.

After that a long beep is sound it means instrument is connecting to the PC.

Data Acquisition window will appear, click on the file & select a new method file, click on Instrument  Parameters view, and feed the instrument parameter as per STP.

then click on the pump set the flow rate and pressure after that click on detector select D2 lamp And set the wavelength to leave all as default.

After that set column oven temp, then select Autosampler to click on the detect rack.

Mode before and after aspiration and rinse dip time (5 sec).

then click on auto-purge select A, B, C, D, Mobile phase click on autosampler fill the time then click on download.

Then go to file click on save method file as and save the file. Then click on Auto purge & wait for Auto purging completion, show progress bar will become 100%.

After completion of the auto purging open the method file and download the method. After baseline correction run the sample.

After a run of sample go-to post-run browse the data and integrate the peaks record the area response of peaks.

Buy: Hplc column storage cabinet for 60 column

HPLC Calibration Procedure:

Frequency: Half Yearly/ after each maintenance. After D2 lamp changed perform detector performance only.

i) Remove the column from the system and replace with a dead volume connector

ii) Flush the system with hot water (50 to 600C) for about half an hour using all channels at a flow rate of 2ml/min using the Following composition

Channel A (25%), B (25%), C (25%) and D (25%)

iii) Carry out the flowing check to test and calibrate HPLC system.

  • Pump performance
  • Injector performance
  • Detector performance
  • Column Oven temperature

Pump Performance

Flow rate accuracy:

a) Remove the column and put all the channel in the reservoir of water, set the flow rate at 1.0ml/min perform in duplicate.

b) Similarly select a flow rate of 2.0ml/min and 3.0ml/min perform in duplicate.

c) Collect the mobile phase at the detector outlet in a dry 10ml volumetric flask and note the time taken to fill the volumetric flask till to the mark perform the test in duplicate.

d) Calculate the corresponding flow rate and record the data in flow rate datasheet.

e) Acceptance criteria ± 2% of set value.

Flow rate consistency:

i) Calculate the % RSD for the retention time obtain in the chromatograph with injection volume 10µl and 20µL from step of injection performance.

ii) Acceptance criteria % RSD NMT 1.0

Compositional accuracy (Gradient Profile):-

(1) Remove the column from the system and replace it with dead volume connector.

(2) Prepare 0.025%V/V solution acetone in water.

(3) Flush the channels for 20min at flow rate 1ml/min using the composition as given below.

TIME(MIN)%WATER (channel A&B)0.025% AcETONE in aqueous (CHANNEL C & D)

(4) Check the compositional accuracy of the HPLC system with the condition given below flow rate 1ml/min

UV at 254nm


Injection delay Time:- 15 minute

(5)Run the gradient using channel composition A, C and B, D

(6) Inject minimum volume of water and record the gradient profile.

(7) Acquired the data till 28 min completion.

(8) Print the overlap plot of gradient profile of combination A, C and B, D.

Acceptance criteria:

(i) Difference in absorbance NMT 0.01 AU

(ii) Difference in time NMT 20 second.

(iii) The gradient profile of A, C, and B, D should overlap each other.

Delay Volume:-

(a) Review the gradient perform under section C compositional accuracy

(b) Note down the time in minutes the actual change in absorbance has taken.

(c) The delay volume of the system can be calculated (in terms of ml) as a time of actual change in absorbance minus 4 minutes.

(d) Acceptance criteria:- the delay volume of system should be NMT 4.5ml.

Injector Performance

Injector Precision:-

(1) Accurately weigh about 100mg of Caffeine in a 100ml volumetric flask containing 10ml of mobile phase and make up the volume with the mobile phase, pipette out 10 ml and further dilute to 100 ml.

(2) Inject 6 times with injection volume of 10µl with the following chromatographic conditions:

  • The column used as per recommended
  • Mobile phase –Water: Methanol (40:60)
  • Flow rate – 1ml/min
  • Injection volume – As per requirement
  • Detector – 272nm (UV)
  • Run time – 6.0 min

(3) Repeat step no. varying injection volume 20µl and 100µl.

(4) Calculate % RSD for data generated each injection volume.

(5) Acceptance Criteria: % rsd NMT 1%.

Injection Linearity:-

(a) Plot regression curve with injection volume on X-axis and mean response of 6 injections on y-axis.

(b) Acceptance criteria:- R square value NMT 0.9990

Injection accuracy:-

(1) Purge auto injector with HPLC grade water.

(2) Fill a standard vial with HPLC grade water and seal with a cap and weigh this vial, record weight (W1).

(3) Programmed  HPLC system for a flow rate and run time of 1min.

(4) Inject 20µl from vial and repeat it for 18 times. After completion of 18 injections, remove the vial and weigh again (W2).

(5) Calculate the average volume (in µl) injected per injection using formula as below:-

W1-W2/18×1000=mg of water/ injection = µl / injection

(6) Record the data in data sheet.

(7) Acceptance criteria:- average volume of injection (µl/injection) should be 20±0.4µl.

Detector Performance:-

Detector Linearity:-

(i) Set up the HPLC system using chromatographic condition given in section (Step no of injection performance at wavelength 254 nm)

(ii) Accurately weigh about 100mg of Caffeine in a 100ml volumetric flask containing 10ml of water and make up the volume with water. Further dilute accordingly with water to get a solution having a concentration of about 10ppm, 15ppm, 25ppm, and 30ppm solution.

(iii) Inject the mobile phase as blank .

(iv) Inject 10ppm, 15ppm, 25ppm, and 30ppm solution of caffeine prepared above in duplicate.

(v) From the data obtain plot a graph of mean area count in Y-axis v/s concentration in X-axis and calculate the value R-square NLT 0.9990. Record the data in the datasheet.

Wavelength accuracy:-

(1) Replace the column with a low dead volume connector.

(2) Prepare 0.01mg/ml solution of caffeine dilute with water Chromatographic conditions:-

  • Flow – 1ml/min
  • Stop time – 6.0min
  • Injection volume – 20µl
  • Mobile phase – Methanol: water (60:40)

(3) Inject the sample at different wavelengths at 202nm, 203nm, 204nm, 205nm, 206nm, 207nm, 271nm, 272nm, 273nm, 274nm, and 275nm.

(4) Acceptance criteria:- 1stMaxima 205±2nm

                                          2ndMaxima 272±2nm

Record the observation in data sheet.

Detector Noise:-

(1) Calculate the baseline noise or the standard deviation of the noise as applicable in the instrument software from section (step no. in detector performance).

(2) Record the observation in data sheet.

(3) Acceptance criteria:- The baseline noise or three times of standard deviation of noise should not be more than 100 microvolt’s and the RSD of baseline noise should not be more than 33%.

Column Oven temperature :-

Set the oven  temperature 250C and wait for achieve the temperature. Check the temperature and note down with the help of external calibrated thermometer. Repeat the procedure by keeping the temperature at 500C and 600C respectively. Difference between thermometer and instrument indicates the temp accuracy.

Sample cooler  temperature :-

(i) Set the sample cooler temperature 50C and wait to achieve the temperature. Check the temperature and note down with the help of an external calibrated thermometer. Repeat the procedure by keeping the temperature at 10°C.

(ii) Acceptance criteria: – ±20C of set temperature.

(iii) Record the observation in data sheet.

Carry over test:

(1)Prepare the chromatograph as describe in Point no. 5.5.1.

(2) Prepare the caffeine sample solution 0.5 mg/ml in methanol.

(3) Inject the 20 µl blank as methanol, caffeine solution and again blank solution.

(4) Observe the area of caffeine solution and second blank solution.

(5) Calculate the carry forward caffeine in blank solution by the formula described in the current version of the format.


Operating Mannual.

 Safety Precautions

(1) Do not use flammable spray near this instrument. they could ignite and cause a fire.

(2) Do not place a heavy object on the power cord. and keep the hot items away it could be damaged resulting in a fire.

(3) Always wear goggles when handling solvents, if the solvents get into the eyes. Blindness could result.


The analytical results depend on the feedback and input instructions feed in the HPLC system, right and correct input instructions are possible with a valid HPLC Calibration Method. Every part installed in HPLC should be calibrated with the proper method and based on validation plan documentation during every HPLC calibration Method. HPLC system totally depends on the precision followed during the analytical process and the calibration is done on frequency. Performing the calibration of HPLC every part of HPLC should be addressed accordingly to the calibration procedure. This machine is very prominent in the pharmaceutical industry in terms of the accuracy of the analysis.

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Darshan Singh

The author has 20 years of experience in the Pharmaceutical Manufacturing Companies. Have exposure to all audit documents for WHO-GMP, PIC/S, USFDA, Export documents, US-FDA documents, Validation of Pharma machinery, and Quality Control Instruments. Expert in Pharma Plant Setup and sale and acquisition of running pharma company.