UPLC calibration procedure: How to Perform

UPLC calibration Description

The following parameters are performed in the UPLC calibration

• Pump calibration
• Injector calibration
• Detector calibration
• Gradient calibration
• Delay volume
• Column oven calibration
• Vial location calibration
• Gradient calibration test for quaternary system

Reagents and chemicals required for calibration

• Uracil standard (with valid certificate of analysis)
• Propyl paraben standard (with valid certificate of analysis)
• Thermometer
• Column: C18, (50×2.1)mm, 1.7µm
• Stopwatch
• water HPLC grade
• Methanol (gradient grade)
• Balance (with valid calibration details)

UPLC calibration frequency

Once in a 6 months ± 5 days

UPLC calibration procedure

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 0.2ml/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 A2 (for binary system) and B1and B2 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 1µl, 2µl, 4µl, 8µl and 10µl of 10 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.

Injector precision calibration

Prepare 20mcg/ml of Uracil solution. Inject 1µl of this solution six times and note down the area response of each injection. Find out the RSD.

Acceptance criteria: The RSD should be less than 1%

Detector calibration procedure

Chromatographic condition

• Column: C18, (50×2.1)mm, 1.7µm
• Flow rate: 0.2ml/minute
• Mobile phase: Water:Methanol (60:40)
• Injection volume: 1µl
• Run time: 2.5 minutes

Wavelength accuracy of UV detector

Prepare 20mcg/ml of Uracil solution. Inject 1 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 1 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

Detector linearity test

Refer Injector precision calibration

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 1µl 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 UPLC column and connect the union in place of column. Flush all the channels first with water and then with methanol. Put the channel A1 in solvent B and channel B1 in the solvent A. Perform the gradient calibration as described in the gradient table:

Solvent A: = 5.6 mg/litre of Propyl paraben in methanol

Solvent B = Methanol

Flow rate: = 0.5ml/minute

Wavelength = 254 nm

Run time: 3 minutes

Inject µl of methanol and record the chromatogram. There should be only two peaks.

Record the start time (in minutes) of the first peak and calculate the delay volume by using the following formulae:

Delay volume = Flow rate x (start time of the first peak – Flow rate)

Calculate the delay volume for the combinations A1/B2, A2/B1, and A2/B2 and record the data

Calculate the average delay volume for all the combinations. It should be in the range of 0.05 to 1.0.

Acceptance criteria: Not more than 0.1ml

Abbreviations:

• UPLC: Ultra-fast liquid chromatography
• C18: Octadecylsilane

References:

• In-house
• IP