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Calibration Procedure / SOP for Ultra Performance Liquid Chromatography (UPLC) System. UPLC can be regarded as a new direction for liquid chromatography. UPLC refers to ultra-performance liquid chromatography, which improves in three areas: “speed, resolution, and sensitivity”.

This Procedure is applicable for Waters Make UPLC – Aquity, UPLC – H Class, Thermo Make U-HPLC Systems, etc.

Procedure (SOP) for UPLC System Calibration

1.0   Calibration Procedure

• • Start-Up of UPLC System

• • Ensure that the instrument is started up (as per the SOP)

• • Ensure that all the units of the system are in working conditions.

2.0   Calibration frequency of UPLC System

• • Following UPLC Calibration Parameters shall be performed Quarterly (± 7 days)

• • Pump by flow-rate accuracy measurement in UPLC System.

• • Pump by flow-rate linearity measurement in the UPLC system.

• • Calibration of Binary pump by gradient flow measurement in the UPLC system.

• • Calibration of PDA/UV-VIS detector by wavelength in UPLC system                                    

• • Noise Calibration for the detector in the UPLC system.

• • Following UPLC Calibration Parameters shall be performed six monthly. (± 15 days)

• • Calibration of PDA/UV-VIS detector for linearity measurement.

• • Autosampler calibration by linearity measurement.

• • Calibration of Autosampler by carryover check.

• • Calibration of Sample cooler and Column oven. (External calibration)

• • UPLC Calibration should be carried out after the following maintenance.

• Pump Calibration of UPLC System :

• • UPLC – Pump Calibration by Flow Rate Accuracy Measurement.

• • Ensure that, the instrument is ready for calibration, and the Start-up procedure is followed.

• • Connect the restricted capillary in place of column connections.

• • Keep the mobile phase in the respective reservoir.

• • Purge the system for each Solvent Line (A1, A2, B1, B2) with filtered HPLC grade water and allow running about 15 min prior to check the flow rate.

Related: Calibration of HPLC System

• • Ensure that the tubing from the reservoir to column inlet shall be free from the air bubble and system pressure should be constant.

• • Take 5 ml clean and dried volumetric flask and keep at the restricted capillary’s outlet in such a way that when the pump is on the water drops fall into a volumetric flask.

• • Set the pump A ( Select Solvent line A1 or A2) for 0.2 ml/minute flow rate, start the pump, and allow running about 5-10 min. Then keep the flask at restricted capillary’s outlet and then simultaneously start the stopwatch.

• • Immediately stop the stopwatch when the volume of water reached the mark in the volumetric flask.

• • Derive the time taken and make an entry in the observation table.

• • Repeat the same procedure for the flow rate of 0.5 ml/min and 1.0 ml/min in 5 ml volumetric flask and record the observations in the given table.

• • Repeat all the steps for Pump B (Select Solvent line B1 or B2). Compare the results for its compliance against limits given in the calibration template.

• • UPLC – Pump Calibration by Flow Rate Linearity Measurement.

• • Ensure that, the UPLC instrument is ready for calibration, and the Start-up procedure is followed.

• • Set up the instrument as per the chromatographic condition mentioned in the calibration format.

• • Ensure that, the column is conditioned before injecting the sample.

• • Use Water: Acetonitrile (90:10) as a mobile phase.

• • Fill vials with sample solution and place them into the sample tray. Vial position is 1:A,2.

• • Select the pump A, Solvent Line A1, or A2.

• • Inject 5µl of sample solution (25ppm Caffeine in water) at 0.6, 0.4, 0.2 & 0.1 ml/min flow rate. Set the run time 2.0 min, 2.0 min, 5.0 min and 10.0 min, respectively for above mentioned flow rate.

• • Record the retention time (RT) for caffeine peak.

• • Record the 1/RT of the Caffeine peak in the observation table.

• • Plot the curve of Flow rate v/s corresponding 1/RT of caffeine peak.

• • Find out the Correlation coefficient “r²” for caffeine peak at four flow rates and record in the calibration template. It should not be less than 0.990.

• • Follow the same procedure for Pump B, Solvent Line B1, or B2.

• • UPLC – Calibration of Binary pump by Gradient flow measurement.

• • If the column is attached to the instrument, remove it, and connect the union.

• • Take the HPLC grade Methanol and use it as a Mobile phase A for Pump-A (Use solvent line A1).

• • Take 2.5mg Propylparaben in 500ml methanol and use as a Mobile phase B for Pump-B (Use solvent line B1).

• • Set the chromatographic condition and Binary gradient flow program as mentioned in the calibration template.

• • Inject 1.0µL of HPLC grade methanol in five replicates and record chromatograms.

• • Record the peak height in the observation table.

• • Plot the curve for % Peak height v/s mean of observed peak height (µv).

• • Find out the %RSD of five readings of peak height and record in the calibration template.

• • Find out the correlation coefficient “r²” from % peak height v/s peak height (µv) graph and record in the calibration template. It should be not less than 0.990.

• • Repeat the same procedure putting solvent line A2 and solvent line B2 in Mobile phase A and Mobile phase B, respectively.

• PDA/UV-Vis Detector Calibration in UPLC System :

• • UPLC – Calibration of PDA/UV-Vis Detector by Wavelength accuracy measurement.

• • Set the chromatographic condition as mentioned in the calibration template.

• • Use Water: Acetonitrile (90:10) as a mobile phase.

• • Use 25ppm caffeine solution in water as a sample solution.

• • For UV Detector, Inject 5µl sample injection for each wavelength from 201nm to 209nm and from 266 to 276nm by the increment of 1nm wavelength.

• • Take the printout of all chromatograms.

• • Find the highest area from both ranges and record the wavelength of the highest area from the respective range of 201nm to 209nm and 266nm to 276nm.

• • The maximum area should be at 273nm ± 2nm for 266nm to 276nm range and should be at 205nm ± 2nm for 201nm to 209nm range.

• • Calibration of PDA/UV-Vis Detector by Linearity measurement.

• • Set up the instrument as per the conditions.

• • Ensure that the column is conditioned before injecting the sample.

• • Use Water: Acetonitrile (90:10) as a mobile phase.

• • Inject the different five concentrations of caffeine samples in duplicate and record the area of the principal peak in the observation table.

• • The five concentrations of caffeine in water are 5ppm, 10ppm, 25ppm, 50ppm, and 75ppm.

• • Plot a linearity curve of concentrations Vs corresponding mean area, using the least square method. Calculate the correlation coefficient, r, and record the observations in the given table.

• • The correlation coefficient should not be less than 0.990.

• • Noise calibration for PDA/UV-Vis Detector.

• • Set the chromatographic condition of UPLC as mentioned in the calibration template.

• • Use Water: Acetonitrile (90:10) as a mobile phase.

• • Run the blank injection in the system for 15min runtime selecting Immediate sample from functions.

• • Collect the baseline noise from 3 min to 15 min at 30sec time interval.

• • The average peak to peak noise from 3 to 15 min at 30sec interval is reported in micro AU.

• • The acceptance criteria: The average peak to peak noise from 3 to 15 min at 30sec interval should be less than 60µAU.

• UPLC – Autosampler Calibration:

• • Calibration of Autosampler by Linearity measurement in UPLC System.

• • Set the chromatographic condition as mentioned in the calibration template.

• • Use Water: Acetonitrile (90:10) as a mobile phase.

• • Ensure that the column is conditioned before injecting the sample.

• • Use 10ppm caffeine in water as a sample.

• • Fill four vials of the sample solution and place it into sample tray at vial position 1: 3, 1:11,  1:19  & 1:27.

• • For 10µl capacity loop, Inject five replicate injections of 2µl, 4µl, 6µl, and 8µl from vials at above-mentioned locations.

• • Record the area response of caffeine peak from chromatograms in the observation table in the calibration template.

• • Calculate the %RSD from five replicate injections for each injection volume and record in the observation table. Determine the correlation coefficient, r². It should not be less than 0.995.

• • UPLC – Calibration of Autosampler by Carryover check

• • Set the chromatographic condition as mentioned in the calibration template

• • Use Water: Acetonitrile (90:10) as a mobile phase.

• • Ensure that the column is conditioned for about 30min with the mobile phase.

• • Inject 5 ml water as blank-1

• • Use 1000ppm caffeine in water as a sample.

• • After getting the baseline from the blank, inject 5µl of the sample in duplicate and record the chromatogram.

• • Again inject 5ml water as blank-2. Observe for any peak at the retention time of the principal peak of caffeine.

• • Record the area of caffeine from the sample and blank solution in the observation table in the calibration template.

• • Calculate the % carryover using the formula,

                                                                                 Area of Caffeine in Blank-2

                                         % Carryover = ——————————————————- x 100

                                                                    Mean Area of Caffeine in Standard Solution

• • The % Carryover should not be more than 0.2%.

• Calibration of Column Oven and Sample cooler.

• • Carry out the calibration for temperature point at 30°C, 50°C and 85°C using digital temperature indicator.

• • Set the temperature of the column oven at 30°C, keep the temperature probe in the center of the column oven, and allow to stabilize the set temperature for at least 15 to 20min.

• • Check the displayed temperature of the column oven and temperature indicator device.

• • Also, carry out for 50°C and 85°C temperature calibration in the same manner and note all readings in the observation table.

• • Set the temperature of the sample cooler at 4°C, keep the temperature probe in the center of the sampler cooler, and allow to stabilize the set temperature for at least 15 to 30min.

• • Check the displayed temperature of the sampler cooler and temperature indicator device.

• • Also, carry out for 10°C and 25°C temperature calibration in the same manner and note all reading in the observation table.

• • Verify the observed temperature v/s actual temperature as per the criteria mentioned in the calibration template.

• • The acceptance criteria for the column oven temperature is ± 2°C of the set temperature and for sample, cooler is ± 4°C of the set temperature.

Precautions during Calibration of UPLC system:

• • Do not start the UPLC system without solvent or mobile phase in the line flow line

• • Ensure that the mobile phase is freshly prepared.

• • Ensure that the mobile phase, diluent, washing solvent, standard, and Samples are filtered through a 0.22 mm filter.

• • Before starting the analysis, ensure all solutions placed properly like seal wash, Weak Needle wash, and Strong needle wash solution.

• • Always switch OFF the detector after the analysis is over and during column washing.

• • Before and after completion of the analysis, the analyst shall wash the column with appropriate solvents and keep the column to its designated place.

• • The analyst shall make an entry of instrument usage and column usage in the Instrument Usage Log and Column Usage Log respectively.

• • In case of noticing any malfunctioning, inform to Head QC or designee.

• • Always flush the system with Appropriate solvent or Water after completion of the analysis.

• • Do not run the system without the inline filter.