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

Objective

To provide a written procedure for the operation of a High-Performance Liquid Chromatography system.

Scope

This SOP covers the operation of the 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 the “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 the “PURGE” key again.

Setting the Flow Rate:

Press the‘ MANUAL SET’ key in the cursor shown on the display. Press the ‘ENTER’ key to select flow enter the required flow rate with a numerical key and press the ‘ENTER’ key to confirm the flow rate.

Setting gradient system:

• Press the ‘INT’ key and the ‘SET PROG’ key to display the shown gradient system program 1 to 9, Select the program and press the ‘ESCAPE’ key to return to the monitor screen without changing the setting.
• Select the program, if the program modifies, Select ‘0’ by pressing the ‘0’ numerical key, or prepare a new program, Select ‘1’ by pressing the ‘1’ numerical key. Press the ‘ENTER’ key to select the program.

• Press the ‘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, and measurement wavelength with a numerical key and an ‘ENTER’ key.
• Correct the input numeric by using the ‘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, and immediately press the ‘START/STOP’ key to start the gradient system pump. If stop the gradient system presses the ‘START/STOP’ key to stop the gradient system pump.
• If the required column temperature is attached the column oven sets the 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.

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Setting Wavelength:

• Set the wavelength range by pressing the ‘WAVELENGTH’ key, enter the required wavelength with the numerical key, and press the ‘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 modifies, Select ‘0’ by pressing the ‘0’ numerical key, or prepare a new program, Select ‘1’ by pressing ‘1’ numerical key. Press the ‘ENTER’ key to select the program.
• Set the time program, and measurement wavelength with a numerical key and an ‘ENTER’ key.
• Correct the input numeric by using the ‘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, and immediately press the ‘START/STOP’ key to start the time program for the detector. If stop the time program press the ‘START/STOP’ key to stop the time program.

Check the Lamp Energy

• Press the ‘CONFIDENCE’ key, Confidence menu is displayed on the screen. Select the ‘CHECK’ menu by pressing the numerical ‘2’ key. Display lamp energy and wavelength accuracy on the display.

RI Detector

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

Fluorescence Detector

• Switch on the power switch provided on the 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 the 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, The Winchrom main screen is displayed.

To Create a New Method File

• Single-click the ‘METHOD’ icon on Winchrom's main screen. The method dialog box is displayed on the screen.
• Choose the 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 ▼.

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Select HPLC and click on details, display show HPLC parameter dialog box.

• Select the temperature for Oven temperature.
• Enter the value for the mobile phase.
• In the mobile Phase enter the solvent name for A, B, C, and D phases.
• In the column field, enter Description, Guard column, I.D., etc.
• In the injection field, enter the loop Size in µl. Select the mode of operation manual or Auto.
• Click on “Program” and enter the flow rate.
• Choose OK to do or choose cancel to abort the dialog box and return to the 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 the Method dialog box, Time Events dialog box display, feed the required parameters then click on OK.
• Select and click Component Table from the Method dialog box, Component Table dialog box display, feed the retention time, and component name, and then click ‘OK’.
• After adding all parameters in the Method parameter, save the method from the file menu. Enter or Select the path and full file name.
• After completion of this procedure inject the solution and injector move in inject position.
• If print of graph is required, click the ‘REPROCESS’ icon in the Win-chrom main screen, select and Open the data file from the file menu with the 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 the file menu and click the print

Column Flushing 

• After completion of analysis clean the column with the following solvents as mobile phase.
• For reverse phase columns:

1. Flush with the same mobile phase, which was needed for analysis for 15 minutes with a 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 needed 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 a 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 a 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 the reverse phase. Store the normal phase column in n-Hexane.

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

Safety Precaution

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

Routine Maintenance

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

Annexure 

Annexure – 1 HPLC Usage logbook

Revision History

Nil

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