SOP for Operation of Shimadzu HPLC (Prominence – i LC – 2030)

OBJECTIVE

To lay down a procedure for the operation of the HPLC system using Lab Solution software. 

SCOPE

This is applicable for the operation of the HPLC system using Lab Solution software for Shimadzu Prominence – i LC – 2030 HPLC system. 

RESPONSIBILITY

It is the responsibility of the QC Officer. 

ACCOUNTABILITY

Executive – Quality Control 

Manager –  Quality Assurance

PROCEDURE

Operation of High-Performance Liquid Chromatography

• Before starting the analysis, ensure that the HPLC System is placed on a clean and dust-free platform.
• Ensure that the instrument has been connected to the power supply. Ensure that all communication cords between the units are connected properly.
• Switch on the instrument (Including Injector, Pump, Column Oven, and UV lamp) by clicking the “ON” button on the extreme left middle corner of the screen and the screen will be changed from red to green. Initialization will occur for 3 - 5 minutes; a self-checkup of the instrument will occur.
• Ensure that the appropriate solvent mixture (water: Methanol) (70:30, v/v) is pumped for seal wash.
• Ensure that the outlet tube from the detector is dipped into a waste bottle.

• Connect the column specified in the individual monograph in the column oven compartment in the flow direction marked on the column. Initially, wash the column by flushing with water: Methanol (70:30) or appropriate solvent if specified in the monograph which is filtered through a 0.45 µ filter for at least 30 minutes.
• Prepare the mobile phase as mentioned in the monograph of a specific product. Fill it in the reservoir bottle. Suspend the inlet tube into the reservoir bottle and ensure that the filter is below the level of the mobile phase.
• After the completion of self-diagnosis, the main menu of the workstation is displayed on the HPLC Screen.
• Double-click the Lab Solution icon on the desktop screen to open the Lab Solution software & Logging in.
• Click on Instrument an HPLC – 2030 a Click on Instrument ON at down assistant Bar.

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Procedure for Method Creation:

• Click on Instrument HPLC – 2030. Go to File a New Method File a Create Method as per Monograph a Download & Close a File a Save Method File As a Save in Specific Product Folder.
• Open the purge valve of the instrument and purge the corresponding solvent line(s) in which the mobile phase has been placed by setting the flow rate of the pump as 1.0 ml/min for a few minutes until the solvent line is completely filled with the new mobile phase.
• Ensure that no air bubbles are noticed in the solvent line.

Procedure for Batch Creation:

• Click on Main at Left Assistant Bar à Real Time Batch a Go to File a New Batch File à Input Data a File à Save Batch File As a Save in Specific Product Folder.

Procedure for Single Run:

• Click on Acquisition on the Left Assistant Bar Start Single Run Input Require Detail Press OK Run the Method & Screen change green to dark blue.

Procedure for Sequence Run:

• To Run a Batch, Click on Main at the Left Assistant Bar a Real-Time Batch a Start Real-time Batch
• The instrument runs automatically after some time.
• While the sequence is running, the chromatogram can be viewed online from the post-run.

Procedure for Data Analysis:

• Click on the Data Explorer sub-window, & double-click on the specific chromatogram.
• Click on “Edit” to edit each parameter value.
• Click on “Integration”. Input Width 5 sec & Slope 200 uV/min.
• Click on “View” to perform processing on the data, & the processing results are displayed in the Chromatogram view and result view – peak table.
• Click on “Edit”. Click Quantitative. For the quantitative method select ‘External Standard’.
• Select 1 for # of calibration Levels.
• Select Conc. for X axis of the calibration Curve.
• Click on “Compound”. Selecting the retention time cell, & clicking the peak in the chromatogram view automatically enters the retention time of that peak to the currently selected [Ret. Time] cell. The retention time can be set by simply clicking the mouse.
• Click on ‘save’ from the top assistant bar.
• Click on ‘Apply to Method’ to save the method file. Tick on ‘current setting’ & ‘Data processing parameters’.
• Click ‘OK’ to save the new data processing parameters.
• Click on the Batch sub-window & open a particular batch. In the method file column add new data processing parameters method & fill down. Save batch.
• Select batch & click on Start. Data processing will start.
• Click on the “Report Format” icon. Open report format. The final report is displayed according to the report style selected.
• Adjust the scale of the chromatogram if required by selecting chromatogram properties. The window of the chromatogram properties will have an option for time scale and chromatogram scale which are adjusted as required. Click OK to close this window.
• Save any changes made in report format. Click on the Print icon at the top of the screen to print the chromatogram. To view the next chromatogram, drag and drop other chromatograms and take a printout similarly.

Procedure for System Shutdown:

• After completion of the analysis, wash the column by downloading the washing method & keep it back in the relevant place.
• Click on ‘Instrument ON’ at the down assistant Bar to disconnect the instrument from the Pump, Lamp, and column oven.
• Close the panel by clicking on the file & then exit.
• Switch off the small round button & switch off the mains of the system controller. After completion switch off the PC.
• Enter the column & instrument usage details in the logbook.

ABBREVIATIONS

HPLC - High-Performance Liquid Chromatography 

UV - Ultra Violet 

PC - Program Controller

ANNEXURE

Nil

REVISION HISTORY
Nil

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