To lay down the procedure for operation of HPLC (Waters alliance with PDA) configuration.
1.0 PURPOSE:
1.1 To lay down the procedure for operation of HPLC (Waters alliance with PDA) configuration.
2.0 SCOPE:
2.1 This SOP is applicable for operation of HPLC (Waters alliance with PDA) 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.
5.3 Connect the required column as per standard test procedure for the sample analysis and column shall be equilibrated by using water: methanol (50:50)/water: acetonitrile.
5.4 Prepare the prescribed mobile phase as per sample testing procedure and fill the reservoirs.
5.5 Before starting of the analysis total system lines washed with mobile phase minimum 15 minutes
5.6 In system monitor select the any parameters by using the ” ?,?, ?,? “ moving keys.
5.7 Before purging the lines set 100% of each composition one by one.
5.8 Select the direct function, after that select wet prime, press “OK”
5.9 Maintain needle and plunger seal wash with 60:40 ratio methanol and water in plunger seal wash bottle.
5.10 After completion of wet prime, set the direct flow rate 0.5ml/min by using moving keys.
5.11 Weekly once sonicate the solvent filters with dilute nitric acid flowed by water and total system lines washed with HPLC water and HPLC grade methanol by dry prime.
5.12 Select the water and methanol for all reservoirs and open the purge valve.
5.13 Click on the direct function, select the dry prime.
5.14 After dry prime is over, close the purge valve.
5.15 Switch on the PDA detector and observe the status.
5.16 Switch on the computer.
5.17 Double click on the Millennium icon.
5.18 Millennium window display on the screen.
5.19 To create a new project:
5.19.1 In Millennium window select and double click the configure system.
5.19.2 Configure system window displays on the screen.
5.19.3 Select projects and click ok.
5.19.4 System/administrator – configuration manager window displays on the screen.
5.19.5 In tool bar open file, click new and select the new project
5.19.6 New project wizard window displays on the screen.
5.19.7 Enter the table space.
5.19.8 Activate the audit trail support.
5.19.9 Click next, select owner group and world, administrator.
5.19.10 Click next; select the view filters, custom fields and methods.
5.19.11 Click next, enter the project name and comments, click finish.
5.20 To load an the existing project:
5.20.1 In Millennium screen double click the browse project.
5.20.2 Browse project window displays on the screen.
5.20.3 Select required project and click ok.
5.20.4 Required project window displays on the screen.
5.21 To create a new instrument method:
5.21.1 Open the required project window.
5.21.2 In project window open file, click new and select instrument method.
5.21.3 Click W2690 icon.
5.21.4 Select general,
5.21.4.1 Stroke volume – Auto.
5.21.4.2 Bubble detector – Active
5.21.4.3 Syringe draw rate – Normal
5.21.4.4 Depth of needle – 0.0
5.21.4.5 Chart out - %A
5.21.4.6 Pre column volume – 0.0
5.21.5 Select degas mode, enter degas mode – continuous.
5.21.6 Select flow,
5.21.6.1 Pressure limits; High limit – 5000 PSI, Low limit – 0.0 PSI.
5.21.6.2 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.
5.21.7 Select temperature
5.21.7.1 Column temperature required, activate the column temperature.
5.21.8 Select solvents, enter the solvents.
5.21.9 Select the 996 PDA icon.
5.21.10 Select general:
5.21.10.1 In data acquisition, enter start wavelength and end wavelength.
5.21.10.2 Sampling rate –1, resolution – 1.2.
5.21.10.3 In instrument control, activate the auto exposure and interpolate 656 nm
5.21.10.4 In analog out puts, activate the analog channel 1 enable.
5.21.11Select channel:
5.21.11.1 In absorbance mode setting, out put – Absorbance
5.21.11.2 Out put wavelength – enter required wavelength
5.21.11.3 Band width – 4.8
5.21.12 In tool bar open file, click save as and enter instrument method name.
5.21.13 Click save.
5.22 Create new processing method:
5.22.11 Open the required project window.
5.22.12 In project window open file, click new and select processing method.
5.22.13 New processing window displays on the screen..
5.22.14 Enter the processing type – LC and click ok.
5.22.15 In integration enter the integration parameters like width, threshold, minimum area and height.
5.22.16 In suitability active the calculate suitability results and enter void volume, select the proper pharmacopoeia.
5.22.17 Select file, click save as and enter the processing method.
5.23 To create a new report method:
5.23.11 Open the required project.
5.23.12 In project window open file, click new and select report method.
5.23.13 New method/group window displays on the screen.
5.23.14 Select create new report method, click ok
5.23.15 Select individual report click next.
5.23.16 Report type- select individual and click finish.
5.23.17 Default report open, select file, click save as and enter the report method name.
5.24 To create a new method set:
5.24.11 Open the required project window.
5.24.12 In project window open file, click new and select method set.
5.24.13 New method set editor window displays on the screen.
5.24.14 Click yes.
5.24.15 Select required instrument method and click next.
5.24.16 Select required process method and required report method, click next.
5.24.17 Enter the method set name, comments and click save.
5.25 To acquire single run:
5.25.11 In project, select tools and click run samples.
5.25.12 2690-996-sys window displays on the screen.
5.25.13 Select proper method set and instrument method.
5.25.14 Click setup, after click the setup screen shows idle.
5.25.15 Press monitor, display the base line screen.
5.25.16 Prepare the samples and open the auto sampler door, put the vials in to carousel, identify the carousel number and vial number.
5.25.17 Create the sequence, open file, click the new sample set method and select empty.
5.25.18 After enter the sample name, vial no., injection volume, number of injections, method set, next injection delay time and channel save the sample set.
5.25.19 After stabilization of base line, select the inject and click the run sample set.
5.25.20 If individual injection enter the method set, injection volume and run time.
5.25.21 In tool bar open inject, click prepare system for injection.
5.25.22 After prepare the injection, open inject and click make injection.
5.26 To reprocess the chromatogram:
5.26.1 Open the project window and select the channels, click update.
5.26.2 Select and double click the required file name.
5.26.3 Review window displays on the screen.
5.26.4 In review window open file menu, click open and select method set.
5.26.5 Select the appropriate method set and click “ok”.
5.26.6 Select the process and click integrate.
5.26.7 After integration completed select the file, choose save and click result.
5.26.8 After save the results close the review window and select the results, click the update.
5.26.9 Select the particular file name
5.26.10 Open tools menu and click print and data enter in to HPLC Logbook.
5.26.11 Back ground processing and reporting window displays on the screen.
5.26.12 Activate the use specified report method and select the required report method and click ok.
5.27 After analysis completed close the project window and log off the millennium and close the detector and computer
5.28 Flush the column with water and methanol for 60minutes each with a flow rate of 1.0ml/min.
5.29 Disconnect the column from the unit after washing and store in an appropriate place.
5.30 Switch off the instrument.
Alliance, hplc waters, equilibrated, instrument method, acquisition
