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SOP for Operation of High-Performance Liquid Chromatography (Merck Model: L7)

Objective
To provide written procedure for operation of High Performance Liquid Chromatography system.

Scope
This SOP covers operation of High Performance Liquid Chromatography system Merck (Model: L7).

Responsibility
QC Officer: Responsible for operation, calibration and maintenance of the instrument as per procedure.
Head of Department: Responsible for calibration and maintenance, timely as per schedule.
QA Officer/QA Manager: Review the records and governing the document.


Procedure
  • Ensure that the instrument is visibly clean and free from dust.
  • Switch the pump power on. Insert the end of the solvent tube into the solvent bottle and open the drain valve.
  • Start the pump with “PUMP ON/OFF” key and press the “PURGE” key. Allow the pump to run until no more air bubbles exit from the outlet. Stop the purging by pressing “PURGE” key again.


Setting the Flow-Rate:
Press the‘ MANUAL SET’ key the cursor show on the display. Press ‘ENTER’ key to select flow and enter the required flow-rate with numerical key and press ‘ENTER’ key to confirm the flow-rate.

Setting gradient system:
  • Press ‘INT’ key and ‘SET PROG’ key display shown gradient system program 1 to 9, Select the program and press ‘ESCAPE’ key returns to the monitor screen without changing the setting.
  • Select the program, if the program modify, Select ‘0’ by pressing ‘0’ numerical key or prepare new program, Select ‘1’ by pressing ‘1’ numerical key. Press ‘ENTER’ key to select the program.
  • Press ‘ENTER’ key, select and add time interval, pump A flow-rate in %, pump B flow rate in %, pump C flow rate in %, pump D flow rate in % with numerical key and press ‘ENTER’ to set value.
  • Set the time program, measurement wavelength with numerical key and ‘ENTER’ key.
  • Correct the input numeric by using ‘CL’ key. Use the ‘DELETE’ key to change the memorized setting numeric.
  • Use the ▲ and ▼ key to be checked program or step of setting program.
  • Load the injection on the injector, immediate press ‘START/STOP’ key to start the gradient system pump. If stop the gradient system presses ‘START/STOP’ key to stop the gradient system pump.

  • If the required column temperature attach the column oven and set required temperature.


UV Detector 
  • Switch on the detector using the POWER switch. After the self-test has passed, a status-indicating screen appears on the display.
  • Pressing the ‘AUTO ZERO’ key, the absorption at the chosen wavelength is set to zero.

Setting Wave length:
  • Set the wavelength range with pressing ‘WAVE LENGTH’ key, enter the require wavelength with numerical key and press ‘ENTER’ key. The wavelength has been set and is indicated on the display.

Setting up the Time Program:
  • Press the‘ TIME PROG’ key to set a time and wavelength for automatic change of measurement wavelength during analysis.
  • Input the program no. to be created or changed. Settable program nos. are from 1 to 9. Pressing the ‘ESCAPE’ key returns to the monitor screen without changing the settings.
  • Select the program, if the program modify, Select ‘0’ by pressing ‘0’ numerical key or prepare new program, Select ‘1’ by pressing ‘1’ numerical key. Press ‘ENTER’ key to select the program.
  • Set the time program, measurement wavelength with numerical key and ‘ENTER’ key.
  • Correct the input numeric by using ‘CL’ key. Use the ‘DELETE’ key to change the memorized setting numeric.
  • Use the ▲ and ▼ key to be checked program or step of setting program.
  • To execute the time program set with the ‘TIME PROG’. Press the ‘START/STOP’ key, a display is provided on the monitor screen and measured values are output.
  • Load the injection on the injector, immediate press ‘START/STOP’ key to start the time program for detector. If stop the time program press ‘START/STOP’ key to stop the time program.


Check the Lamp Energy
  • Press ‘CONFIDENCE’ key, Confidence menu display on the screen. Select ‘CHECK’ menu with pressing numerical ‘2’ key. Display lamp energy and wavelength accuracy on the display.

RI Detector
  • Switch on power switch provided on right side corner of the detector. After the self-test has passed, a status-indicating screen appears on the display.
  • Press ‘SENS’ key to check the sensitivity of detector or add require sensitivity with pressing ‘SENS’ key and then press ‘ENTER’ key to confirm the value.
  • Press ‘FUNC’ key and then press ‘AUTOZERO’ key to detector display will be zero.
  • For purging press ‘FUNC’ key and then press ‘AUTOZERO’ key

Fluorescence Detector
  • Switch on power switch provided on left side corner of the detector. After completion of initialization screen appears on the display for wave length setup.
  • Set the extinction wavelength and emission wavelength by pressing wavelength key on the front panel of the detector.

Software Operation 
  • Start windows on your PC and in the program group ‘Winchrom’, click the winchrom icon, Winchrom main screen is display.
To Create a New Method File
  • Single click the ‘METHOD’ icon in winchrom main screen. Method dialog box has display on the screen.
  • Choose Open or ‘New’ option from the File menu. Display show new Method dialog box on the screen.
  • Add Runtime and Minimum peak width value with ▲ and ▼.


Select HPLC and click on details, display show HPLC parameter dialog box.
  • Select the temperature for Oven temperature.
  • Enter the value for mobile phase.
  • In the mobile Phase enter the solvent name for A, B, C, D phases.
  • In the column field, enter Description, Guard column, I.D., etc.
  • In the injection field, enter the loop Size in ┬Ál. Select the mode of operation manual or Auto.
  • Click on “Program” and enter the flow rate.
  • Choose OK to done or choose cancel to abort the dialog box and return to previous screen.


Select and click Parameters, Method Parameter dialog box display.
  • Click on detector, detector parameter dialog box display, feed the parameters, select detector, and then click on OK.
  • Click Integration, Integration parameter dialog box display, feed the parameters then click on OK.
  • Click Screen, Screen parameter dialog box display, feed the parameters then click on OK.
  • Click Report, Report parameter dialog box display, feed the parameter then click on OK.
  • Click System Suitability, System suitability parameter dialog box display, feed the
  • Click ‘OK’ to confirm all method parameters.

  • Select and click Time Events from Method dialog box, Time Events dialog box display, feed the require parameters then click on OK.
  • Select and click Component Table from Method dialog box, Component Table dialog box display, feed the retention time, component name and then click ‘OK’.

  • After adding all parameter in Method parameter, save method from file menu. Enter or Select path and full file name.
  • After completion of this procedure inject the solution and injector move in inject position.
  • If print of graph is require, click the ‘REPROCESS’ icon in Win-chrom main screen, select and Open data file from the file menu with selecting path. Reprocess with click reprocess in Operation menu. If any changes are require set the require parameters in data file.
  • Select print command from file menu and click the print

Column Flushing 
  • After completion of analysis clean the column by the following solvents as mobile phase.
  • For reverse phase columns:
  1. Flush with the same mobile phase, which was need for analysis for 15 minutes with 1.0 ml flow rate.
  2. Flush with water for 30 minutes.
  3. Then flush with Methanol for 15 minutes at a flow rate of 1.0 ml / minute.


  • For normal phase columns :
  1. Flush with the same mobile phase which was need for the analysis for 15 minutes at a flow rate of 1.0 ml / minute.
  2. Then flush with n -Hexane at a flow rate of 1.0 ml / minute.

  • Change over from Reverse phase to Normal phase :
  1. After the analysis is over flush the column with water for 30 minutes, followed by Methanol for 15 minutes.
  2. Remove the reverse phase column (C18 or C8) and attach a dead volume instead of column.
  3. Now flush the system with Acetonitrile followed by Chloroform and n -Hexane (all 15 minutes each).
  4. Remove dead volume and fix a normal phase column (e.g. silica)
  5. Continue flushing for 15 minutes.
  6. Maintain the reverse phase column in Methanol.

  • Change over from normal to reverse phase :
  1. After the analysis is over flush the columns with n- Hexane for 15 minutes.
  2. Remove the normal phase column (C18 or C8) and attach a dead volume instead of column.
  3. Flush the system with Chloroform followed by Acetonitrile and Methanol for 15 minutes.
  4. Now fix the reverse phase column and flush the column for 15 minutes
  5. Continue the analysis in reverse phase. Store the normal phase column in n -Hexane.

  • Enter the details in HPLC usage logbook as per given in Annexure –1.

Safety Precaution
After analysis run the system washes with water for 30 minutes if buffer solution used in
mobile phase followed by Methanol for 15 minutes.

Routine Maintenance
  • Clean the instrument by dry cloth.
  • Clean the system with hot water without connecting the column. Than Methanol and finally with water.
  • Sonicate the suction filter with 2 M Nitric acid.

Annexure 
Annexure – 1 HPLC Usage logbook

Revision History
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