SOP for Operation of HPLC Waters Alliance with Empower

PURPOSE:

To lay down the procedure for the operation of HPLC (Waters alliance with UV) with Empower.

SCOPE:

This SOP is applicable to the Quality control / Stability department.

RESPONSIBILITY:

Assistant – QC / Stability to operate the HPLC.

ACCOUNTABILITY:

Manager - Quality control / Stability To ensure the compliance for established procedure

PROCEDURE:

Procedure For General Cleaning

• Check that the power supply to the instrument is switched ‘OFF’ before cleaning.
• Clean the instrument with a clean dry cloth every day and occasionally wet cloth dipped in dilute soap solution may be used. Precaution to be taken to clean the instrument immediately with dry cloth to remove the moisture.

Preparation of Mobile Phase

• Always use filtered (through 0.45-micron membrane filter) mobile phase/solvents.

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Operation of Waters 2695 Separation Module

• Switch on the waters 2695 by pressing the on/off switch to the symbol ‘I’ (on) position (the on/off switch is located on the left side of the module Initialization takes place and the Ready state is displayed.
• Keep all A, B, C, D channel tubing as well as seal wash and needle wash tubing in to methanol
• Press the “Develop Method’s” key to access the prime and purge all the lines, and select “ Auto start Plus”.
• Press the “ run “ key to prime and purge all the lines with methanol.
• After completion of priming of all channels remove the required tubing from the bottle and keep it back in the respective bottles.
• Perform the wet priming in the case of the Agilent 1100 series hooked to Empower software as given below.
• Access the instrument method editor click the quaternary pump to display its tabs, and click the flow tab.
• In the Flow %A, %B, %C, and %D cells, enter 100% for the first solvent you want to use for priming.
• Open the purge valve and press ‘Apply’.
• After completion of the prime stop flow in the instrument method editor and then close the purge valve.

Note: The purge valve cannot be closed without stopping the flow in the instrument editor (in software). Since the high flow rates will create high backpressure on the pump motor due to the column connection.

• Repeat the steps 7 to 10 when solvents/solutions are changed.

Procedure to Operate 2487 Detector

• Switch on the waters 2487 detector by pressing the on/off switch to the I (on) position (the on/off switch is located at the right-hand side bottom in the front). Initialization takes place.
• The lamp will glow after 5 minutes of warm-up time.
• Self-diagnosis goes on.
• The screen will display the earlier wavelength and the absorbance.

Procedure to Operate Empower

• Power up the system instrumentation in the following order.
• Chromatographic instruments (wait until all internal calibration and diagnostic routines are complete before powering up the next instrument).
• Peripheral devices connected to the Empower system, such as a printer, client, etc,
• The Windows desktop appears once the computer is powered up asking to log on to the operating system.
• Press the “Alt+Ctrl+Delete” buttons simultaneously. Windows logon screen appears.
• Enter the User Name, Password, and Domain Name (as Empower2” in User Name, Password, and Domain text boxes respectively to log on to the “Empower” domain.
• Click on “Login…” Button. Empower login windows appear. Enter the allotted User name of the individual, and password of the individual, and select the database as “WAT5.EMPOWER2.COM”(Default) from the dropdown list and click on OK to log on to Empower.
• Double-click the “Browse project” button, then existing projects will appear to select the required project from the project menu and double-click to open the project.
• Go to “Tools” on the menu bar on the top and select Quick set. or press the Quick Set button on the toolbar.
• The “Select System” window is displayed. Select the required system (Acquisition) and click OK.

• The quick set will start initializing. The quick set window is displayed.
• Select the required instrument method; from the “Instrument Method” window at the bottom.
• Click “Edit” to open the Instrument method. The ”Instrument Method” edit window is displayed.
• Click on the w2690/95 separation module icon, and separation module parameters are displayed
• Verify general system parameters in the general button. The parameters are stroke volume “auto”, and bubble detect? marked, syringe draw rate (µl/sec) is “Normal”, pre-column volume ( µl)“ 0.0 ”, Depth of needle ( mm) is “0.0”, Chart out is “%a”.
• Select the degas button and degas mode is “on”. In case sparging is required for any channel, select the flow rate ( ml/min) in the desired channel.
• In case sparging is selected then go to the events button and enable events and feed required parameters for example: set sparge toggle between on and off time, pulse time, etc.
• Press the flow button and enter the pressure limits (lower and upper), In programmed flow choose either isocratic or gradient based on analysis specification.
• Set total flow in ml and 100 % of any one channel. In case of gradient select the percentage of flow from multiple channels.
• Press the temperature button and then enable column temperature if required and set the target temperature in degrees with tolerance limits.
• Select the sample temperature enable the option if cooling is required and set the target temperature in degrees with tolerance limits.
• Feed channel solvent details in the solvents button.
• Click on the detector icon, and detector parameters are displayed.

Operate the Detector in Single Wavelength Mode

• Select the “Single” wavelength mode from O Single O Dual
• Select the “Single” Channel from Single      Dual
• Press the “Channel 1” button.
• Verify the wavelength. Change the wavelength if necessary.
• Go to “File” and select “Save”, to save the changes.
• To save the method with another name, select “Save as” and give the instrument method name in the “Instrument Method” window. Click on Save.
• Close the Instrument Method Editor window by clicking on “X” on the right side top of the window.

ALSO READ: SOP for HPLC System Using Chemstation Software from Agilent

Operate the Detector in Dual Wavelength Mode

• Select the “Dual” wavelength mode from O Single Dual
• Select the “Dual” Channel from Single Dual
• Press the “Channel 1” button.
• Verify the wavelength. Change the wavelength if necessary.
• Press the “Channel 2” button.
• Verify the wavelength. Change the wavelength if necessary.
• Go to “File” and select “Save”, to save the changes.
• To save the method with another name, select “Save as” and give the instrument method name in the “Instrument Method” window. Click on Save.

• Close the Instrument Method Editor window by clicking on “X” on the right side top of the window.
• To perform a single injection; click on “Single” on the Quick set window.
• Enter the information like Vial No., Sample name, Function, Method set, Injection volume, and Runtime in the sample-loading table.
• Open the door of the injector and wait for the carousel to get released.
• Select the carousel and keep the vial to be injected in the carousel (sample loading tray) and load the carousel into the instrument. Close the door.
• Go to “Inject” on the main menu bar on the top and select “Make injection” or press the “Inj” button below the Sample table; to start the injection.
• For performing injections of more than one sample, select “Samples” on the Quick Set Window.
• Fill the “Sample table” by “Auto Fill” mode.
• Select the “Sample Name” field in the Sample table and click on the right button of the mouse. Select “Auto Fill” from the menu.
• Enter information like “Sample Name”, “Incrementing prefix” and “Incrementing suffix”, “Vials numbers to be injected” and click OK.
• Similarly, Autofill is done for the “Method set”, “# of inj”, “inj. vol”, and “Run time” fields by following the above procedure.
• The Sample table is filled with the above information.
• Go to “File” on the main menu bar on the top and select “Save sample set method as”.
• The “Sample set method” window is displayed. Enter the sample set name and press OK to save.
• Select Run mode to “Run only” for data acquisition only.
• Select the Run mode to “Run and process” for acquisition and processing. Select this mode only when the processing method is created earlier.
• Select a Run mode to “Run and report” for acquisition, processing, and reporting. Select this mode only when processing and reporting methods are created and are included in the method set.
• Open the door of the injector and wait for the sample loading into the carousel.
• Fill the carousel with the vials to be injected according to the queue in the sample-loading table.
• Insert the carousel into the instrument and close the door.
• Go to “Inject” on the main menu bar on the top and select “Run Sample set” or press the “Run” button (Green button) on the tools bar.
• “Run Sample Set” window is displayed. Press “Run” to start the sample set.
• The sample in the first row will be highlighted with red color and the injections get started.
• After completion of the injections, click open the project window.
• Click the channel button in the views on the project screen. All the Channels are displayed. Alternatively, select the “All channels by date” filter to view all Channels.
• Select a completed injection in the channel view table.
• Go to “Tools” on the main menu bar on the top and select “Review”. Or click the Start-review button in the tools. Review data: Project window appears.
• The review window is displayed with the chromatogram.
• Go to “Window” on the menu bar on the top and select “Processing method”.
• Enter the parameter's Peak width, Minimum area, Minimum height, and Threshold value in the respective boxes of the Integration table. Program the events.
• Click the “Components” button. Enter “Component name”, “Retention time”, and “RT window”.
• Click on the “Suitability” button and select Calculate the suitability results.
• Enter “Void volume” and select the required pharmacopeia or “All” to calculate System suitability as per all pharmacopeia.
• Close the “Processing Window”.
• Go to “Process” on the main menu bar and select “Integrate” and then “Quantitate”. Or press the “Integrate” button and then the “Quantitate” button on the tools bar.
• Now zoom the chromatogram to check for proper integration of peaks. If the integration is not proper alter the above parameters in the integration event table.
• Go to “File” and select “Save method as”.
• Enter the file name and click OK.
• Click File and ‘Save Result’. Then file ‘Exit’.
• Click the Display results button in the View of the Project window.
• Select the above-saved result of the sample in the Result view table.
• Click the Preview button in the tools and select the required report or select the report method from the Preview: Report Method Selection.
• The instrument starts creating the report and is displayed on the screen.
• Double-click on the chromatogram to modify the chromatogram scaling.
• Select User defined in scaling type.
• Feed required scale parameters, like X-start, Y-start, X-end, and Y-end. Double-click the Peak result table below the auto-scaled chromatogram to modify the table format by selecting the required parameters.

• Select the required peak results by double clicking the parameters like Component name, retention time, area, area %, height, height % in the peak fields, Channel name, Date acquired, Injection from the Sample fields System suitability parameters like USP Tangent, USP Tailing, Resolution from system suitability fields.
• Click the file and save.
• Click the Printer button to print the current report.
• Click File Exit.
• Go back to the Quick Set control window and select Run and Report mode against square to enable the continuous printing of the results after completion of the subsequent injections.
• Enter the analysis log book.

ALSO READ: SOP for ThermoFisher Scientific HPLC

Creation of New Method Set

• After browsing the project and selecting the system, go to the File menu and select the new method.
• In New Method select the Instrument method. Select the instrument system from the list and click OK.
• Detector parameters are displayed.
• Select the wavelength mode, Single or Dual channel.
• Enter the required wavelength in the corresponding Channel.
• Go to the File menu and click save as and give a name to it.
• Go to the File menu and select New Method.
• In New Method select the processing method.
• Select LC on the screen and click OK.
• In the processing method screen, select the Integration and feed the parameters peak width, threshold, minimum area, minimum height, etc.
• Go to the component table and enter the required details like Name, R.T, R.T window, etc.
• Go to Suitability and enable the system suitability option and select the fields required.
• Go to the File menu and click save as and give a name to it.
• Go to the File menu and select New Method.
• In New Method select the Reporting method
• In the displayed screen select the required format and click next.
• In the displayed screen select the required format and click Finish.
• Print preview will have appeared.
• Go to the File menu and click save as and give a name to it.
• Go to the system screen and go to Edit, select New method set By using the wizard for method set creation select Instrument Method created and click next.
• Feed the created Processing method and Reporting methods and click next.
• Give the Method name and click finish.

Sequence of Closing Operation

• Exit the Quick Set control screen by clicking ‘File-Exit’.
• Exit the project screen by clicking ‘File-Exit’.
• Exit the Empower-session manager screen by clicking the logout arrow.
• Log out from Windows and Switch off the computer and monitor.

ABBREVIATIONS

Inj – injection

Inj. Vol-injection volume

µl – microliter

mm- millimeter

ml- milliliter

ANNEXURE

Nil

REVISION HISTORY

Nil

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