UPLC System Calibration Procedure

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.Waters 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.

Name of Module Type of Maintenance Calibration required
Pump
Change of plunger assembly
Flow rate accuracy measurement.
Detector Change of lamp / optical lens The linearity of the response check and wavelength accuracy check.
Autosampler Change of syringe/sample loop / Rotor Carryover check and Linearity/Injection accuracy by weight measurement.
Column oven/Cooler Replacement of sensor Calibration of column oven.
Sample cooler Replacement of sensor Calibration of sample cooler.
  • 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.

pharmabeginers

Janki Singh is experienced in Pharmaceuticals, author and founder of Pharma Beginners, an ultimate pharmaceutical blogging platform. Email: [email protected]

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