SOP Title-Operation of High Performance Liquid Chromatography (HPLC)

SOP Title-Operation of High Performance Liquid Chromatography (HPLC)

 

• OBJECTIVE

To lay down a procedure  for  operation of High Performance Liquid Chromatography.

• SCOPE

This SOP is applicable for operation of High Performance Liquid Chromatography located at Quality Control Department.

MAKE: Shimdhzu

 * RESPONSIBILITY

• QC personal shall be responsible for implementation of
• QC Head – shall be responsible for compliance of this procedure.

 

• PROCEDURE :
  • Operation Procedure :
    • Switch “ON” the mains power supply.
    • Switch on the instrument.
    • Initialization the Instrument.
    • Wait for few minute to initialization the instrument.
    • Instrument screen display.
  • Rom check ——————   ok
  • Ram Check ——————   ok
  • Conditioning ——————  ok
  • Seeking Home —————— ok
  • Checking Wave —————— ok
  • Checking Energy—————— ok
  • User Name —————— Admin
  • Login Pass-ID-No——————00000
  • D₂ Lamp Energy ——————-on
  • Press Five Times (0) Key for the (Log) Screen.

The Screen Display OK, Use the (F2) Key, For Display Menu.

Then Press the (F5) Key For Display Method On System Screen.

• Software Operation: Start the Computer “LC solution” icon Display on Desktop.
  • Setting Analytical Conditions:
    • Double Click “LC Solution” icon on the Desktop.
• Click Instrument 1 or 2 or 3 or 4 for Real Time Analysis.
• Enter User ID and Password then Click OK.
•   Go to file →New method File.
• Click Data Acquisition in Toggle Assistant Bar located in the left side of the Window and Click the Instrument Parameter icon.
• Maximize the Instrument Parameters View window. Here we can select either normal mode or advance mode.
• Enter the analytical conditions in the respective modules and click Download.
• The save as window will open and give the name for Method. Select the path for the particular project  in this project method, data and batch name should come .Method name having the extension of *.lcm.
• Minimize the Instrument Parameter Window.
• Check the LC Status whether it is in Ready mode or not .If it is in Ready mode, System is Ready for sample Analysis.
• Creating Single Run:
  • Check the baseline stability in the chromatogram window for UV detector, click Acquisition →Plot Start. After checking the baseline, Acquisition → Plot top.
  • Click Data Acquisition in Toggle Assistant Bar located in the left side of the Window.
  • Single Run Window will open. Enter the sample name and Sample ID.
  • Current Method will open automatically otherwise open the required method.
  • Enter the Data File name and select auto increment if required.
  • Select the Background file for background correction and enter the Data Description for reporting purpose.
  • Enter the set vial number, tray number and injection volume of the
  • Click Advance for selecting the report format file.
  • Select Report and open the required report template.
  • Click ok and click Start to start the run.
• Creating Batch:
  • Click the Window → Show Window →Batch Table.
  • Select File → New Batch.
  • Right Click the mouse and select Table Easy setting.
  • Select Batch Table: New for New Batch and Append for add the sequence With exiting batch.
  • Select Standard →Enter the Starting Vial Number and Ending Vial number, Enter the tray, enter the Repetition, and Enter Injection Volume and Data Name. Follow the same for Unknown.
  • All the Information will be set in the batch tab.
  • Change or modify the necessary parameters such as Sample ID, Sample Name, Injection Volume, Vial Number, Tray No etc.,
  • File →Save Batch File As and enter the batch name in the respective Project
  • Here the Batch name having the extension of *.Icb
  • Click Batch Start in the Toggle Assistant Bar to start the sequence.
• Processing of Data:
  • Click the data Analysis icon in the Toggle Assistant Bar.
  • That data analysis window will open in the Post run Analysis Window.
  • Open the data from the Toggle Data Explorer bar.
  • Click Analyze icon in the Toggle Assistant Bar. Data Analysis Window Will open.
  • In Integration Tab, enter or check the necessary width, slop, drift value.
  • In Performance Tab, enter the calculation method, unretained peak time and column length. Click  Apply and OK.
  • Click Wizard icon from the Toggle Assistant Bar.
  • Compound Table Wizard 1/5 window will open. Check the necessary width, slop, drift value.
  • Click the Program icon in this table. Integration time program window will Open along with the current data name. Do the necessary integration and click Stimulate and OK.
  • Right click the mouse in the chromatogram window and select the manual Integration tool bar for addition integration icon.
  • Click Next to go for Compound Table Wizard 2/5.Click the select column in this window for naming the peak and click Next for Compound Table Wizard 3/5.
  • Enter the number in the # of calibration Levels, select fit type linear, enter the unit in this window and click Next for Compound Table Wizard 4/5.Check the necessary parameters in the window and click Next for Compound Table Wizard 5/5.

• Enter the peak name in the Name column in this window and give the concentration of the Samples in the conc1, conc2…column and click Finish.
• Right click the mouse in the chromatogram window and select Proper ties. Select the required peak top commands like area, RT, height etc.,
• Click View in the compound Table view. Click Apply to Method in the Toggle Assistant Bar. Save Method As window will open.
• Click Save and Click Yes to over write the method name. Select Method Parameters Window will open.
• To know the run table, to know RT, Area, Height etc., select Result in the compound Table view. To add more parameters right click the mouse in this table and select Table Style, double click the parameters which located in the right side of the window.

• Report Generation:
• Click the Top icon in the Toggle Assistant Bar and select the Report Format icon.

*  Blank Report Format will open.

• Select Item →Common→ Sample Information. Draw a square in the report format.
• Select Item →LC/PDA Common→ Chromatogram. Draw a square in the report format.
• Select Item →LC/PDA Common→ Peak Table. Draw a square in the report format.
• File →Save Report Format File As→ Give the name of the Report Format.
• To Add or modify any parameter, Double click the mouse in the respective filed and the necessary changes and then Save the report format. Open the data and drag the data and drop in the report format filed to get the report.
• To take the print outs of chromatograms and its report table: (Batch Run)
  • Create a report as mentioned in the above mentioned procedure.
  • After proper integration of peaks, open the Batch.
  • Check the Report Output column and open the report format file in the Report format file column.
  • Process the Batch for printouts.
• Washing Procedure:
  • Reverse Phase Chromatography:
• Off the D2 Lamp and wash the column with water about 30 Minutes.
• Then wash the column with mixture of water and strong organic solvent, used in mobile    phase in (50: 50) ratio about 30 minutes.
• Store the column in acetonitrile or in methanol.
• Normal Phase Chromatography:
• Wash the column with strong solvent used in mobile phase.
• Store the column in isopropyl alcohol.
• Procedure for Shutdown the Instrument:
  • Before shutdown the system, rinse the auto sampler at suitable solvents And purge the pump.
  • Switch off the system sequentially system and controller.
• Cleaning:
  • Daily clean the instrument, computer with clean and dry lint free cloth to remove the dust.
  • Before switching off the instrument Rinse and Purge the Auto injector with Water: methanol(50:50) mixture.
  • Clean the plunger head by flushing water through the cleaning tubes.
  • Sample Vials: Open the cap. Discard contents of vials. Rinse the vials and     caps with tap water. Then rinse with mild detergent solution. Finally rinse with purified water with tap water. Then rinse with mild detergent solution. Finally rinse with purified water and dry the vials in the Glass-ware drying oven.

 

• ABBREVIATION(s)

Abbreviation	Full Description
SOP	Standard Operating Procedure
QA	Quality Assurance
QC	Quality Control
HPLC	High Performance Liquid Chromatography
RT	Retention Time
ID. No.	Identification Number

 

*Note – Ready to use SOP available in “DOWNLOAD” Section.