Calibration of HPLC: How to Perform

Calibration of HPLC: Acceptance Criteria Summary

Calibration of HPLC

The following parameters are performed in HPLC calibration

• Pump calibration or flow rate accuracy
• Injector calibration
• Detector calibration
• Vial location calibration for (auto sampler)
• Gradient calibration
• Drift and Noise

Reagents and other requirements: Calibration of HPLC

• Uracil standard (with valid COA)
• Propyl paraben standard (with valid COA)
• Thermometer
• C18 column, (150×4,6)mm, 5µm
• stop watch
• water HPLC grade
• Methanol (gradient grade)

HPLC calibration frequency

Once in a 6 months ± 5 days

Flow rate accuracy or pump calibration procedure

The flow rate accuracy of the pump can be evaluated simply by the time required to collect a predetermined volume of the eluent at different flow rate setting. Use distilled water as a mobile phase. drain the tubing line with water to ensure that there are no air bubbles trapped in the line.

Set the flow rate of the pump at 0.5ml/minute and start the pump. Allow the pump to run for 10 minutes. Keep 5 ml of class A volumetric flask below the outlet and start the stopwatch. Stop the stop watch when the bottom of the meniscus reaches the 5 ml mark on the flask. Weigh the mobile phase in the gram and calculate the volume of the mobile phase by dividing it by the density of the mobile phase.

Volume = Mass or Weight/Density

Record the elapsed time in seconds. In the same way repeat the process for channel B (for binary system) and B, C and D for quaternary system. Calculate the flowrate using the following formula:

Flow rate = (Volume of the flask/Measured time in seconds)/60

Set the flow rate 1 ml/minute, 2ml/minute for 10 minutes, similarly the mobile phase using 10 ml and 20 ml of class A volumetric flasks respectively. Weigh the mobile phase in the gram and calculate the volume of the mobile phase by dividing it by the density of the mobile phase. Record the result.

Acceptance criteria ±2% of the flow rate

Injector calibration procedure

Inject 5µl, 10µl, 20µl, 50µl and 100µl of 20 mcg/ml solution of Uracil. Record the area response of each injection. Draw the linearity plot between area response and injection volume.

Acceptance criteria: The correlation coefficient should not be less than 0.999

Note: Chromatographic condition as given in the Detector calibration will be considered.

Detector calibration procedure

Chromatographic condition

• Column: C18, (150×4.6)mm, 5µm
• Flow rate: 0.9ml/minute
• Mobile phase: Water:Methanol (60:40)
• Injection volume: 20µl
• Run time: 5 minutes

Wavelength accuracy of UV detector

Prepare 20mcg/ml of Uracil solution. Inject 20 µl of this solution at wavelengths 250 nm, 252 nm, 254 nm, 256 nm, 258 nm, 260 nm, 262 nm, 264 nm, 266 nm and 268 nm. and note down the area response.

Acceptance criteria: The wavelength maxima should be 258 nm ± 2nm

Wavelength accuracy of PDA detector

Prepare 20mcg/ml of Uracil solution. Inject 20 µl solution of Uracil in the wavelength range between 200 nm to 400 nm. Extract the chromatogram at 250 nm, 252 nm, 254 nm, 256 nm, 258 nm, 260 nm, 262 nm, 264 nm, 266 nm and 268 nm. and note down the area response.

Acceptance criteria: The wavelength maxima should be 258 nm ± 2nm

Injector precision calibration

Prepare 20mcg/ml of Uracil solution. Inject 20 µl it six times and note down the area response of each injection. Find out the RSD.

Acceptance criteria: The RSD should be less than 1%

Vial location calibration (for auto sampler)

Prepare 20mcg/ml of Uracil solution. Take 5 different HPLC vial and fill each vial with this solution. Keep the each vial at different locations in the autosampler. Inject 20 µl from each vial. Record the area response.

Acceptance criteria: The RSD of area response should be less than 1%

Gradient calibration for Quaternary system

Disconnect the HPLC column and connect the union in place of HPLC column. Flush all the channels first with water and then with methanol. Put the channel A and channel B in the solvent B and channel C and channel D in the solvent A, Perform the gradient calibration as described in the gradient table:

• Solvent A: = 5.5mg/litre of Propyl paraben in methanol
• Solvent B = Methanol
• Flow rate: = 2ml/minute
• Wavelength = 254 nm
• Run time: 18 minutes

Gradient table

Note: The above gradient calibration is for water system. These may be some changes in the other system.

Use the following formula to calculate height of the peak A/B/C and A/B/D:

% A/B/C = (Height of A/B/C ÷ Height of A/B/C/D full scale)x100

% A/B/D= (Height of A/B/D ÷ Height of A/B/C/D full scale)x100

There should be only three peaks. Name the peaks as full scale A/B/C and A/B/D. Record % peak height of all three peaks. % height of the peak A/B/C and A/B/D should be 10% ±5 of the full scale peak. Record the data.

Acceptance criteria: 10% ±5

Drift and Noise

Drift and Noise is the Change in baseline and these occur due to various factors like temperature, vibration, solvent quality etc.

The following chromatographic condition is used to calculate the drift and noise:

• Column: Use union in place of column
• Mobile phase: Use distilled water
• Flow rate: 1.0ml/minute
• Injection volume: oµl (Use air or empty vial)
• Wavelength: 254 nm
• Column temperature: 25 to 40^oC
• Run time: 30 minutes

Procedure: Inject air in the above chromatographic condition and calculate the drift and noise

Acceptance criteria:

• Noise should be less than or equal to 60 micro AU for UV detector and less than or equal to 80 micro AU for PDA detector
• Drift should be less than 10 milli AU/Hr for UV detector and less than 10 milli AU/Hr for PDA detector

Carryover calibration

Use the following formula to calculate the carryover:

% Carryover = (carryover area in blank/test area)x100

Acceptance criteria: Not more than 0.01%

You may also want to check out other articles on my blog, such as:

• How to calibrate KF?
• How to calibrate GC?
• How to calibrate a UV spectrophotometer?
• How to calibrate FTIR?
• How to perform calibration of pH meter?
• How to calibrate KF apparatus?
• How to perform verification of analytical balance?
• How to calibrate UPLC?

Interview Questions on HPLC Calibration

2. How often should HPLC calibration be performed?

Answer:
Calibration frequency depends on several factors, including the complexity of the analysis, system usage, and regulatory requirements. Generally, HPLC systems should be calibrated:

• Before using the system for new method development or critical analysis.
• Regularly, typically every 6 months for routine monitoring.
• After maintenance, repairs, or any part replacement (e.g., pump seals, columns).
• After significant changes, like new column installation or solvent changes.

Always follow specific guidelines outlined by the manufacturer and regulatory bodies (e.g., FDA, ICH).

3. What are the key parameters to check during HPLC calibration?

Answer:
Key parameters to check during calibration include:

• Flow rate accuracy (Pump calibration).
• Injection precision (Injector calibration).
• Retention time (to check system stability).
• Resolution and peak symmetry (to evaluate column performance).
• Detector response (Detector calibration).
• Gradient accuracy (for gradient-based separations).
• Baseline drift/noise (to ensure detector reliability).

These parameters ensure optimal performance and reproducibility of results.

4. How is the linearity of the HPLC detector calibrated?

Answer:
The linearity of the HPLC detector is typically calibrated by preparing a series of standards with known concentrations of the analyte. The detector response (e.g., absorbance, fluorescence) is plotted against concentration, and a calibration curve is constructed. The curve should show a linear relationship (R² ≥ 0.99). Deviations from linearity can indicate issues with the detector, such as non-linearity at high concentrations or detector saturation.

5. What is the significance of system suitability tests (SST) in HPLC?

Answer:
System suitability tests are predefined performance criteria that must be met before starting a series of analyses. These tests include evaluating parameters such as:

• Retention time consistency.
• Resolution between peaks.
• Tailing factor (peak symmetry).
• Column efficiency (number of theoretical plates).

SST ensures that the HPLC system is operating within acceptable limits for precise and reliable results, and is particularly important for regulatory compliance.

6. How do you calibrate the HPLC temperature (oven) control?

Answer:
HPLC column temperature is critical for consistent retention times and separation. To calibrate the temperature:

• Use a calibrated thermocouple or precision thermometer to measure the actual temperature inside the column oven.
• Compare the measured temperature with the oven’s setpoint.
• Adjust if necessary to ensure ±2°C of the set temperature.

Temperature control should be verified regularly, especially if there is a noticeable shift in retention times or changes in baseline stability.

7. What should be done if an HPLC system shows high carryover between samples?

Answer:
High carryover (residual analyte remaining from a previous sample) can be addressed by:

• Regularly cleaning the injector and other parts exposed to sample solutions.
• Ensuring wash protocols are set up to adequately clear the system between injections (e.g., longer rinse times, higher solvent volumes).
• Checking the injector precision and calibration to ensure accurate volumes are delivered.
• Reducing sample concentration in cases where carryover is due to saturation of the injector.

Abbreviations:

• HPLC: High-performance liquid chromatography
• C18: Octadecylsilane

References:

• In-house
• IP

Further Reading

• <621> Chromatography