To lay down the procedure for operation of HPLC
1.0 PURPOSE:
1.1 To lay down the procedure for operation of HPLC (Waters 600E connected with 2487 detector/Waters 1525 with 2487 detector) configuration.
2.0 SCOPE:
2.1 This SOP is applicable for operation of HPLC (Waters 600E with 2487 detector/Waters 1525 with 2487 detector) 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 600 E pump/1525 pump.
5.3 Connect the required column as per standard test procedure for the sample analysis and column shall be equilibrated by using either water: methanol or water: acetonitrile (50:50) ratio.
5.4 Prepare the mobile phase as per standard test procedure and fill the reservoirs.
5.5 Before starting to the analysis total system lines purge with buffer solution or with mobile phase (if isocratic analysis).
5.6 Set the flow by using Home button on pump monitor.
5.7 Set the flow rate and enter the gradient compositions if applicable.
5.8 Open the drain valve of the pump for purging purpose.
5.9 Pump purging with mobile phase.
5.10 After purging close the drain valve of the pump.
5.11 Switch on the 2487 detector and observe the status.
5.12 Switch on the computer.
5.13 Double click on the Millennium icon.
5.14 Millennium window display on the screen.
5.15 To create a new project:
5.15.1 In Millennium window select and double click the configure system.
5.15.2 Configure system window displays on the screen.
5.15.3 Select projects and click ok.
5.15.4 System/administrator – configuration manager window displays on the screen.
5.15.5 In tool bar open file, click new and select the new project
5.15.6 New project wizard window displays on the screen.
5.15.7 Enter the table space.
5.15.8 Activate the audit trail support.
5.15.9 Click next, select owner group and world, administrator.
5.15.10 Click next; select the view filters, custom fields and methods.
5.15.11 Click next, enter the project name and comments, click finish.
5.16 To load an existing project:
5.16.1 In Millennium screen double click the browse project.
5.16.2 Browse project window displays on the screen.
5.16.3 Select required project and click ok.
5.16.4 Required project window displays on the screen.
5.17 To create a new instrument method:
5.17.1 Open the required project window.
5.17.2 In project window open file, click new and select instrument method.
5.17.3 Click W2487 icon.
5.17.4 Select general.
5.17.4.1 Channel 1 – Enable
5.17.4.2 Description – Product name with required wavelength, nm.
5.17.4.3 Press Channel-1, enter wavelength required & sampling rate required -1 pt/sec.
5.17.4.4 Data mode – Absorbance-Ch1
5.17.4.5 Filter type – Hamming, Time constant – 1.0 sec.& ratio minimum:0.100 AU.
5.17.4.6 Press Auto Zero, then click ?on wavelength change, ?on inject change, ?on from key pad or event change
5.17.4.7 Click on Analog-A icon, Analog output mode as Absorabance ch-1, AUFS – 2.0, polarity +, absorabance set-0.00, voltage offset (mv), ?on chart mark enable.
5.17.5 Select degas mode, enter degas mode – continuous.
5.17.6 Select 600E Icon/1525 Icon,
5.17.6.1 Click pump type 600E/1525, head volume 100µl.
5.17.6.2 Pressure limits; High limit – 5000 PSI, Low limit – 0.0 PSI.
5.17.6.3 Programmed flow; Select required pump mode (Isocratic/gradient), in isocratic enter the total flow, %A is 100%, if it is gradient enter the flow program as per the test method.
5.17.7 Select temperature
5.17.7.1 Column temperature required, activate the column temperature.
5.17.8 Select solvents, enter the solvent names as used in method.
5.17.9 In tool bar open file, click save as and enter required instrument method name.
5.17.10 Click save.
5.18 Create new processing method:
5.18.1 Open the required project window.
5.18.2 In project window open file, click new and select processing method.
5.18.3 New processing window displays on the screen..
5.18.4 Enter the processing type – LC and click ok.
5.18.5 In integration enter the integration parameters like width, threshold, minimum area and height.
5.18.6 In suitability active the calculate suitability results and enter void volume, select the proper pharmacopoeia.
5.18.7 Select file, click save as and enter the processing method.
5.19 To create a new reporting method:
5.19.1 Open the required project.
5.19.2 In project window open file, click new and select report method.
5.19.3 New method/group window displays on the screen.
5.19.4 Select create new report method, click ok
5.19.5 Select individual report click next.
5.19.6 Report type- select individual and click finish.
5.19.7 Default report open, select file, click save as and enter the report method name.
5.20 To create a new method set:
5.20.1 Open the required project window.
5.20.2 In project window open file, click new and select method set.
5.20.3 New method set editor window displays on the screen.
5.20.4 Click yes.
5.20.5 Select required instrument method and click next.
5.20.6 Select required process method and required report method, click next.
5.20.7 Enter the method set name, comments and click save.
5.21 To acquire single run:
5.21.1 In project, select tools and click run samples.
5.21.2 Select proper method set and instrument method.
5.21.3 Click setup, after click the setup screen shows idle.
5.21.4 Press monitor, display the base line screen.
5.21.5 After stabilization of base line, stop the run (select the inject and click the stop run).
5.21.6 Enter the method set, injection volume and run time.
5.21.7 In tool bar open inject, click prepare system for injection.
5.21.8 After prepare the injection, open inject and click make injection through
Rheodyne injector.
5.22 To process the chromatogram:
5.22.1 Open the project window and select the channels, click update.
5.22.2 Select and double click the required file name.
5.22.3 Review window displays on the screen.
5.22.4 In review window open file menu, click open and select method set.
5.22.5 Select the appropriate method set and click “ok”.
5.22.6 Select the process and click integrate after feeding the peak width, threshold.
5.22.7 After integration completed select the file, choose save and click result.
5.22.8 After save the results close the review window and select the results, click the update.
5.22.9 Select the particular file name
5.22.10 Open tools menu, click print and enter into HPLC logbook.
5.22.11 Back ground processing and reporting window displays on the screen.
5.23 Activate the use specified report method and select the required report method and click ok.
5.24 After analysis completed close the project window and log off the millennium and close the detector and computer
5.25 Flush the column with water and methanol for 60minutes each with a flow rate of 1.0 ml / min.
5.26 Disconnect the column from the unit after washing and store in an appropriate place.
5.27 Switch off the instrument.
Liquid chromatograph, waters, power supply, millennium icon, filters
