To lay down the procedure for operation of HPLC (Shimadzu).
1. OBJECTIVE
1.1 To lay down the procedure for operation of HPLC (Shimadzu) -equipment identification number: LAB3
2. RESPONSIBILITY
2.1 Sr.Assitant-QC
2.2 Jr.Executive/Sr.Executive-QC
3. ACCOUNTABILITY
3.1 Assist.Manager/Designee-QC
4. PROCEDURE
4.1 Connect the instrument to 230 volts A/C power supply.
4.2 Switch on the instrument and pumps.
4.3 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 or ratio.
4.4 Prepare the prescribed mobile phase as per testing procedure and fill the reservoirs.
4.5 Open the drain valve of the pumps and press purge button to flush the lines with mobile phase.
4.6 After purging close the drain valve of the pump.
4.7 Set the pressure control to the maximum by using func key.
4.8 Set the flow rate initially 0.2 ml/min using the func key on the pump.
4.9 Press pump key to start the pump.
4.10 By increasing the flow rate slowly with 0.2ml increment up to required flow rate.
4.11 Switch on the UV- detector.
4.12 Enter the required wavelength by using the numerical keys after self-calibration of detector.
4.13 Switch on the waters bus SAT/IN module and computer.
4.14 Double click on the Millennium icon.
4.15 Millennium window display on the screen.
4.16 TO CREATE A NEW PROJECT
4.16.1 In Millennium window select and double click the configure system.
4.16.2 Configure system window displays on the screen.
4.16.3 Select projects and click ok.
4.16.4 System/administrator – configuration manager window displays on the screen.
4.16.5 In tool bar open file, click new and select the new project
4.16.6 New project wizard window displays on the screen.
4.16.7 Enter the table space.
4.16.8 Activate the audit trail support.
4.16.9 Click next, select owner group and world, administrator.
4.16.10 Click next; select the view filters, custom fields and methods.
4.16.11 Click next, enter the project name and comments, click finish.
4.17 TO LOAD THE EXISTING PROJECT
4.17.1 In Millennium screen double click the browse project.
4.17.2 Browse project window displays on the screen.
4.17.3 Select required project and click ok.
4.17.4 Required project window displays on the screen.
4.18 TO CREATE A NEW INSTRUMENT METHOD
4.18.1 Open the required project window.
4.18.2 In project window open file, click new and select instrument method.
4.18.3 Select General.
4.18.3.1 Description – Enter the required description.
4.18.3.2 Sampling rate – 1
4.18.3.3 Units - mµ
4.18.3.4 Sampling factor-1000
4.18.3.5 Bcd polarity – (-)
4.19 CREATE NEW PROCESSING METHOD
4.19.1 Open the required project window.
4.19.2 In project window open file, click new and select processing method.
4.19.3 New processing window displays on the screen..
4.19.4 Enter the processing type – LC and click ok.
4.19.5 In integration enter the integration parameters like width, threshold, minimum area and height.
4.19.6 In suitability active the calculate suitability results and enter void volume, select the proper pharmacopoeia status.
4.19.7 Select file, click save as and enter the processing method.
4.20 TO CREATE A NEW REPORT METHOD
4.20.1 Open the required project.
4.20.2 In project window open file, click new and select report method.
4.20.3 New method/group window displays on the screen.
4.20.4 Select create new report method, click ok
4.20.5 Select individual report click next.
4.20.6 Report type- select individual and click finish.
4.20.7 Default report open, select file, click save as and enter the report method name as required.
4.21 TO CREATE A NEW METHOD SET
4.21.1 Open the required project window.
4.21.2 In project window open file, click new and select method set.
4.21.3 New method set editor window displays on the screen.
4.21.4 Click yes.
4.21.5 Select required instrument method and click next.
4.21.6 Select required process method and required report method, click next.
4.21.7 Enter the method set name, comments and click save.
4.22 TO ACQUIRE SINGLE RUN
4.22.1 In project, select tools and click run samples.
4.22.2 LABI/LC-03 window displays on the screen.
4.22.3 Select proper method set and instrument method.
4.22.4 At chromatogram monitor screen click the right side mouse button and select the customize channels.
4.22.5 Customize channels window displays on the screen.
4.22.6 Enter the required wavelength and press apply and ok buttons.
4.22.7 Click setup, after click the setup screen shows idle.
4.22.8 Press monitor, display the base line screen.
4.22.9 After stabilization of base line, stop the run (select the inject and click the stop run).
4.22.10Enter the method set, injection volume and run time.
4.22.11In tool bar open inject, click prepare system or prepare system icon for injection.
4.23 Wash the syringe with methanol and water thoroughly.
4.24 Then flush the loop by injecting water and methanol thoroughly.
4.25 Rinse the syringe thoroughly with sample solution.
4.26 After stable the base line, load the sample in loop and turn the reodine
4.26.1 In LABI/LC-03 window main tool bar open inject, click prepare system or prepare system icon for injection.
4.27 TO REPROCESS THE CHROMATOGRAM.
4.27.1 Open the project window and select the channels, click update.
4.27.2 Select and double click the required file name.
4.27.3 Review window displays on the screen.
4.27.4 In review window open file menu, click open and select method set.
4.27.5 Select the appropriate method set and click “ok”.
4.27.6 Select the process and click integrate.
4.27.7 After integration completed select the file, choose save and click result.
4.27.8 Close the review window.
4.27.9 Press update icon at particular project window.
4.27.10 Select the required file name.
4.27.11 In main tool bar open tools and click print.
4.27.12 Print window displays on the screen.
4.27.13 Select appropriate report method and click ok icon.
4.27.14 After collect the report enter the HPLC logbook as per annexure-1.
4.28 After analysis completed close the project window and log off the millennium and switch of the detector and computer
4.29 Flush the column with water and methanol for 60minutes each with a flow rate of 1.0ml/min.
4.30 Disconnect the column from the unit after washing and store in an appropriate place.
4.31 Switch off the instrument.
5. ABBREVIATONS
HPLC - High performance liquid chromatograph
QC - Quality control
6. REFERENCES
Equipment Manual
Millennium software manual
7. ANNEXURES
7.1 Annexure –1 HPLC logbook
Shimadzu hplc, quality control, equilibrated, numerical, browse project, threshold
