SOP for Calibration of HPLC Waters System

PURPOSE

To provide a calibration procedure for High-Performance Liquid Chromatograph (Waters HPLC).

SCOPE

This procedure applies to all the HPLCs (Waters) used in the Quality Control department.

RESPONSIBILITY

It is the responsibility of the HPLC Operating Chemist

Department Head

DOCUMENT REFERENCE

SOPs: Core procedure, Calibration of Equipment

Forms: Calibration of HPLC (Waters) system

SCHEDULE

The calibration of HPLC system shall be performed in the first week of every third month.

PROCEDURE

CALIBRATION OF PUMP:

Accuracy of flow rate:

• Fill filtered water in the pump reservoir and purge the pump.
• Set the Flow rate of the pump to 1.0 ml/minute and ensure no air bubble is stuck up in the path.
• Take a clean 10 ml Class – Graduated cylinder.
• Note the time and immediately start collecting water into the 10 ml graduated cylinder.
• After 9 minutes note the time and remove the 10 ml cylinder.

Acceptance Criteria: The volume of water collected should be 9.0 ml + 0.2.

ALSO READ: SOP for Operation of HPLC Waters Alliance with Empower

Gradient Accuracy check:

Chromatographic Conditions:

Column: Hypersil ODS 100 x 4.6 mm ID, 5µ or equivalent

Flow rate: 2.0 ml /min.

Detector wavelength: 254 nm

Injection volume: 20 µl

Flow rate: 2mL/min

Runtime: 30 minutes

Mobile Phase

Mobile Phase A or C: Water generated by Milli-Q Apparatus

Mobile phase B or D: 0.3%v/v Acetone in filtered water generated by Milli-Q

Preparation of Mobile Phase B: Take 3mL of acetone in 1000mL of water generated by Milli-Q

Gradient program:

Procedure using Pump A and Pump B:

Time (min)	Mobile phase B (%)
0.01	0
2.00	0
2.01	10
8.00	10
8.01	50
13.00	50
13.01	90
18.00	90
18.01	100
23.00	100
23.01	0
23.01	0

• Open the drain valves and purge the flow lines of each channel
• Equilibrate the column with initial concentration with the above-mentioned conditions and wait until the baseline is stable
• Adjust the baseline level to fit the full Scale of the integrator
• Inject exactly 20 µl of mobile phase A and start the time program for the gradient accuracy test.
• Determine the signal level at 0%(B Con),10%(B Con), 50%(B Con), 90%(B Con) and 100%(B Con)
• Calculate the actual B concentration level at 10%(B Con), 50%(B Con), 90%(B Con) Using 0% (B Con) and 100% (B Con)

Calculation:

Calculate the actual B con. level at 10% = 

B concentration level at 10% - B concentration level at 0%

B concentration level at 100% - B concentration level at 0%

Similarly, calculate the actual B con. level at 50%(B Con) and 90%(B Con)

Acceptance Criteria :

At B concentration 10% level should be between 9.0 and 11.0

At B concentration 50% level should be between 49.0 and 51.0

At B concentration 90% level should be between 89.0 and 91.0

Procedure using Pump C and Pump D:

Time (min)	Mobile phase B (%)
0.01	0
2.00	0
2.01	10
8.00	10
8.01	50
13.00	50
13.01	90
18.00	90
18.01	100
23.00	100
23.01	0
23.01	0

• Open the drain valves and purge the flow lines of each channel
• Equilibrate the column with initial concentration with the above-mentioned conditions and wait until the baseline is stable
• Adjust the baseline level to fit the full Scale of the integrator
• Inject exactly 20 ?l of mobile phase A and start the time program for the gradient accuracy test.
• Determine the signal level at 0%(D Con),10%( D Con), 50%( D Con), 90%( D Con) and 100%( D Con)
• Calculate the actual D concentration level at 10%( D Con), 50%( D Con), and 90%( D Con) Using 0% (D Con) and 100% (D Con)

Calculation:

Calculate the actual B con. level at 10% = 

D concentration level at 10% - D concentration level at 0%

D concentration level at 100% - D concentration level at 0%

Similarly, calculate the actual B con. level at 50%(D Con) and 90%(D Con)

Acceptance Criteria:

At D concentration 10% level should be between 9.0 and 11.0

At D concentration 50% level should be between 49.0 and 51.0

At D concentration 90% level should be between 89.0 and 91.0

ALSO READ: Qualification of High Performance Liquid Chromatography

CALIBRATION OF PDA DETECTOR:

Wavelength Accuracy

• Go to diagnostics and click on PDA calibration.
• The screen will appear and feed the expose time as 44 seconds and press the start.
• run icon, followed a spectrum will appear.
• After 1 minute stop the run and press ‘Detect Lines and take out the printout.
• Press Calibration accuracy and note down the variation at 486.0 nm and 656.1 nm.

1. Go for PDA diagnostics click on the ‘lamp’ and ‘wavelength’ icons and press run.
2. Take the printouts of all the above data.

Acceptance Criteria : 

The variation should be within + 1 nm at the specified wavelengths.

Lamp Intensity Test

Measure the lamp intensity from the spectrum obtained from the wavelength accuracy test at 220 nm and record the results

Acceptance Criteria : 

The lamp intensity at 220 nm should be greater than or equal to 5000.

CALIBRATION OF UV DETECTOR:

Wavelength Calibration

• Switch on the UV detector and warm up at least 30 minutes
• Set the Methanol flow 1 mL per minute and wash the detector cell for at least 30 minutes.
• Press the shift switch followed by the calibrate switch and press enter to start the calibration.
• After completion of the calibration process, a message will appear as follows.
• ‘Calibration successful, maximum deviation since last calibration = 0.2 nm.
• (current deviation in nm).

1. Go for wavelength diagnostics and click on lamp and wavelength icons and press run.

Acceptance Criteria: The variation should be within + 1 nm.

Lamp Energy test:

• Set the Methanol flow to 1 mL per minute and wash the detector cell for at least 30 minutes. Press the shift button followed by the diagnostic button twice and then select Sample and reference energy mode feed the 220 nm and press enter to observe the lamp energy at the reference cell.

Acceptance Criteria: The reference energy at 220 nm should be more than 20.

CALIBRATION OF PUMP, AUTOINJECTOR and DETECTOR

Chromatographic system:

Column : Hypersil ODS 100 x 4.6 mm ID, 5µ or equivalent

Flow rate: 1.5 ml /min.

Detector wavelength: 254 nm

Injection volume: 20 µl

Runtime: 8 minutes

Preparation of Calibration Standard:

Weigh accurately about 150 mg each of Dimethyl phthalate, Diethyl phthalate,10mg of Biphenyl, and 30 mg of O-Terphenyl, and transfer into a 100 ml volumetric flask. Dissolve and dilute to volume with methanol.

Procedure:

• Equilibrate the column for 30 minutes with the mobile phase at a flow rate of 1.5 ml/min (with the initial concentration in case of a gradient system).
• Inject exactly 20µl of methanol as blank into the system and record the chromatogram.
• Program the data processor to inhibit the integration of peaks due to blank.
• Inject 20µl of Isocratic Standard 6 times and record the chromatograms.
• Calculate the %RSD for Retention Times observed from the replicate injections to check the precision of the Pump flow rate.
• Calculate the %RSD for areas observed from the replicate injections to check the precision of the Autosampler and Detector.

Acceptance Criteria: % RSD calculated for Retention times should not be more than 1.5%% RSD calculated for areas should not be more than 1.5%

Note :

In case the equipment fails in calibration, call the service engineer to repair the instrument. The data about previous samples analyzed (analyzed after the previous due of calibration) using the equipment/instrument shall be reviewed and the review shall be documented. All the proceedings shall be in accordance to the core procedure, “Calibration of Equipment”.

ANNEXURE

Nil

REVISION HISTORY

Nil

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