To lay down the procedure for operation of HPLC (Agilent) configuration.
1.0 PURPOSE:
1.1 To lay down the procedure for operation of HPLC (Agilent) configuration.
2.0 SCOPE:
2.1 This SOP is applicable for operation of HPLC (Agilent) in Quality control department.
3.0 RESPONSIBILITY:
3.1 Assistant – QC
4.0 ACCOUNTABILITY:
4.1 Executive – QC
5.0 PROCEDURE:
5.1 Connect the instrument to 230 volts A/C power supply.
5.2 Switch on the instrument by pressing switch to turn on the variable wavelength detector, auto sampler, pump and vacuum degasser one by one.
5.3 Connect the required column for the sample analysis and equilibrate for 10 minutes with water/methanol or acetonitrile.
5.4 Prepare the mobile phase as per standard testing procedure and fill the reservoirs.
5.5 Switch on the computer.
5.6 Double click on the Millennium icon.
5.7 Millennium window display on the screen.
5.8 To create a new project:
5.8.1 In Millennium window select and double click the configure system.
5.8.2 Configure system window displays on the screen.
5.8.3 Select projects and click ok.
5.8.4 System/administrator – configuration manager window displays on the screen.
5.8.5 In tool bar open file, click new and select the new project
5.8.6 New project wizard window displays on the screen.
5.8.7 Enter the table space.
5.8.8 Activate the audit trail support.
5.8.9 Click next, select owner group and world, administrator.
5.8.10 Click next; select the view filters, custom fields and methods.
5.8.11 Click next, enter the project name and comments, click finish.
5.9 To load the existing project:
5.9.1 In Millennium screen double click the browse project.
5.9.2 Browse project window displays on the screen.
5.9.3 Select required project and click ok.
5.9.4 Required project window displays on the screen.
5.10 To create a new instrument method:
5.10.1 Open the required project window.
5.10.2 In project window open file, click new and select instrument method.
5.10.3 A1100 window displays on the screen.
5.10.4 Click Quaternary pump icon:
5.10.4.1 Flow:
5.10.4.1.1 Minimum pressure - 0
5.10.4.1.2 Maximum pressure - 5000 psi
5.10.4.1.3 Auxiliary pump
5.10.4.2 Minimum strokes:
5.10.4.2.1 Automatic - activate
5.10.4.2.2 Value (10-6 per bar) - activate
5.10.4.3 Primary channel:
5.10.4.3.1 Automatic - activate
5.10.4.4 Pump mode:
5.10.4.4.1 Activate either isocratic or gradient as per standard test procedure.
5.10.4.4.2 If it is gradient enter the flow rate and flow program.
5.10.4.4.3 If it is isocratic enter the flow rate.
5.10.4.5 Channel
5.10.4.5.1 General
5.10.4.5.1.1 Channel off - activate
5.10.4.6 Solvents - enter the solvents description
5.10.5 Click the auto injector icon:
5.10.5.1 General:
5.10.5.1.1 Syringe volume - Enter the volume as per standard test procedure.
5.10.5.1.2 Seat capillary volume (µl) - 2.3
5.10.5.1.3 Wash vial No. - 97
5.10.5.1.4 Injection mode - injection with needle wash
5.10.5.2 Auxiliary:
5.10.5.2.1 Draw speed (µl/min) - 200
5.10.5.2.2 Draw position (mm) - 0
5.10.5.2.3 Eject speed (µl/min) - 200
5.10.6 Click VW detector icon:
5.10.6.1 General:
5.10.6.1.1 Wave length - enter required wavelength as per standard test procedure.
5.10.6.1.2 Auto zero pre run - on
5.10.6.1.3 Signal polarity - (+)
5.10.6.1.4 Peak width - enter the required peak width range
5.10.6.1.5 Auto zero position - on
5.10.6.1.6 Lamp type - enter lamp type
5.10.6.1.7 Enable analysis when lamp is off - No
5.10.6.1.8 Margin for negative absorbance (mau)- 100
5.10.6.1.9 UV lamp - on
5.10.6.1.10 Analog out:
5.10.6.1.10.1 Analog range - 1.0V full scale
5.10.6.1.10.2 Attenuation (mau) - 100
5.10.6.1.10.3 Zero off set (%) - 5
5.10.6.2 Channel:
5.10.6.2.1 General:
5.10.6.2.1.1 Sample with out reference -channel off
5.10.6.2.1.2 Reference only -channel off
5.10.6.3 Events:
5.10.6.3.1 If it is necessary enter the wavelength program
5.11 Create new processing method:
5.11.1 Open the required project window.
5.11.2 In project window open file, click new and select processing method.
5.11.3 New processing window displays on the screen..
5.11.4 Enter the processing type – LC and click ok.
5.11.5 In integration enter the integration parameters like width, threshold, minimum area and height.
5.11.6 In suitability active the calculate suitability results and enter void volume, select the proper pharmacopoeia.
5.11.7 Select file, click save as and enter the processing method.
5.12 To create a new report method:
5.12.1 Open the required project.
5.12.2 In project window open file, click new and select report method.
5.12.3 New method/group window displays on the screen.
5.12.4 Select create new report method, click ok
5.12.5 Select individual report click next.
5.12.6 Report type- select individual and click finish.
5.12.7 Default report open, select file, click save as and enter the report method name.
5.13 To create a new method set:
5.13.1 Open the required project window.
5.13.2 In project window open file, click new and select method set.
5.13.3 New method set editor window displays on the screen.
5.13.4 Click yes.
5.13.5 Select required instrument method and click next.
5.13.6 Select required process method and required report method, click next.
5.13.7 Enter the method set name, comments and click save.
5.14 To acquire single run:
5.14.1 In project, select tools and click run samples.
5.14.2 2497-sys window displays on the screen.
5.14.3 Connect the required column as per standard test procedure for the sample analysis and column shall be equilibrated by using either water: methanol (50:50) or water: acetonitrile ratio.
5.14.4 Open the purge valve, enter the flow rate at update area and press apply icon.
5.14.5 After two minutes press stop flow icon and close the purge valve.
5.14.6 Again enter the flow rate at up date area and press apply for column equilibration.
5.14.7 After 30 minutes prepare the mobile phase as per standard test procedure and fill in to reservoirs.
5.14.8 Follow the same points 4.14.4 to 4.14.5.
5.14.9 Select proper method set and instrument method.
5.14.10 Click setup, after click the setup screen shows idle.
5.14.11 Press monitor, display the base line screen.
5.14.12 After stabilization of base line, stop the run (select the inject and click the stop run).
5.14.13 Prepare the samples, fill the vials and put in the auto sampler tray.
5.14.14 Run sequence:
5.14.14.1 Create the sequence, open file, click the new sample set method and select empty.
5.14.14.2 After enter the sample name, vial no., injection volume, number of injections, method set, next injection delay time and channel save the sample set.
5.14.14.3 Then select the inject and click the run sample set.
5.14.15 Run individual injection:
5.14.15.1 If individual injection enter the method set, injection volume and run time
5.14.15.2 In main tool bar open inject, click prepare system for injection
5.14.15.3 After prepare the injection, open inject and click make injection.
5.15 To reprocess the chromatogram:
5.15.1 Open the project window and select the channels, click update.
5.15.2 Select and double click the required file name.
5.15.3 Review window displays on the screen.
5.15.4 In review window open file menu, click open and select method set.
5.15.5 Select the appropriate method set and click ‘ok’.
5.15.6 Select the process and click integrate.
5.15.7 After save the results close the review window and select the results, click the update.
5.15.8 After integration completed select the file, choose save and click result.
5.15.9 Close the review window.
5.15.10 Press update icon at particular project window.
5.15.11 Select the required file name.
5.15.12 In main tool bar open tools and click print.
5.15.13 Print window displays on the screen.
5.15.14 Select appropriate report method and click ok icon.
5.15.15 After collect the report enter the HPLC logbook.
5.16 Flush the column with water and methanol for 60minutes each with a flow rate of 1.0ml/min.
5.17 Disconnect the column from the unit after washing and store in an appropriate place.
5.18 Close the Agilent HPLC 1100 window.
5.19 Switch off the computer and instrument.
Wavelength detector, auto sampler, pump, vacuum, millennium icon, logbook
