To lay down a procedure for Operation of HPLC (Shimadzu with LC 10AT and SPD-M10 AVP).
1.0 PURPOSE:
1.1 To lay down a procedure for Operation of HPLC (Shimadzu with LC 10AT and SPD-M10 AVP).
2.0 SCOPE:
1.2 This SOP is applicable for HPLC (Shimadzu with LC 10AT and SPD-M10 AVP) in quality control department.
3.0 RESPONSIBILITY:
3.1 Assistant – Quality Control
3.2 Executive – Quality Control
4.0 ACCOUNTABILITY:
4.1 Manager – Quality Control
5.0 PROCEDURE:
5.1 Connect the instrument to 230 volts A/C power supply.
5.2 Switch on the instrument pumps A and B
5.3 Connect the required column for sample analysis and equilibrated for ten minutes with water/methanol or acetonitrile.
5.4 Prepare the prescribed mobile phase as per sample testing procedure and fill the reservoirs.
5.5 Open the drain valve of the pumps and purge the mobile phase.
5.6 After purging close the drain valve of pump.
5.7 Set the pressure control to maximum and minimum (minimum pressure always set to zero) using function key.
5.8 Set the concentration of pump-A&B in the conc. mode of pump-A (Master pump).
5.9 If the testing program is gradient enter the time events and concentration of pumps in the edit menu of pump-A.
5.10 Press edit key and enter the func key up to display Bcon of the monitor.
5.11 Press enter key and enter the specific concentration.
5.12 Follow the points 5.10, 5.11up to the time program completion
5.13 At the end of the program press the back key up to display of stop in the monitor
5.14 Set the flow rate initially 0.2 mL / min using the func key.
5.15 Press pump key to start the pump.
5.16 By increasing the flow rate slowly with 0.2 mL increment up to required flow rate.
5.17 Switch on the PDA - detector.
5.18 Switch on the computer.
5.19 Double click on class VP main icon.
5.20 Class-vp window displays on the screen
5.21 Select and double click the instrument – 1 icon
5.22 Instrument –1 window displays on the screen.
5.23 Click SPD-M10 AVP lamp icon to glow the lamp.
Note: In case any error massage displays close the detector and restart the computer.
5.24 Create the new method:
5.24.1 In main tool bar open the file menu, click the method and select new.
5.24.2 Untitled method will be loaded
5.24.3 Click the method menu and select the PDA setup.
5.24.4 PDA setup window displays on the screen.
5.24.5 In general enter the parameters i.e., wavelength range, Run time and select the trigger type external.
5.24.6 In multi enter the wavelength, bandwidth, click active and apply.
5.24.7 Open file menu, click the method and select save as.
5.24.8 Enter the particular method name and click ok.
5.25 Loading the existing method:
5.25.1 In main tool bar open file menu, click the method and select open.
5.25.2 Method file window displays on the screen.
5.25.3 Select the required method and click ok.
5.26 Load the preview run:
5.26.1 In main tool bar open control menu and click the preview.
5.26.2 In monitor show method save yes or no, click yes.
5.26.3 Monitor displays base line.
5.26.4 After base line stabilization stop the preview run.
5.26.5 Open control and select stop run.
5.27 Syringe washed with methanol and water.
5.28 Flush loop by injecting water and methanol thoroughly.
5.29 Prepare the sample solution as per sample testing procedure.
5.30 Rinse the syringe thoroughly with sample solution before injecting sample.
5.31 Acquired the single run:
5.31.1 In main tool bar open control and click single run.
5.31.2 Single run window displays on the screen.
5.31.3 Enter the particulars like identification of the sample, file name and click start.
5.31.4 Massage shows waiting for trigger on the screen.
5.31.5 Inject the sample solution in loop and load it.
5.31.6 After loading down the screen massage shows running sample.
5.31.7 The chromatogram shall be displayed on the screen.
5.31.8 After completion of run time automatically stop the single run.
5.32 Integration of the chromatogram:
5.32.1 In main tool bar open the method menu and click integration events.
5.32.2 Integration events window displays on the screen.
5.32.3 Enter the integration parameters such as width, threshold, minimum area, lock on/off etc…
5.32.4 After entering the integration events close the window.
5.32.5 Open analysis and click analyze.
5.32.6 Then chromatogram shall be integrated with peak area.
5.33 Print the chromatogram and peak report:
5.33.1 In main tool bar open reports menu, click view and select the method custom report.
5.33.2 Method custom report window displays on the screen.
5.33.3 Default method custom report open.
5.33.4 Enter the parameters like sample ID, method name, data file name sample description and analyst name.
5.33.5 Double click on default chromatogram.
5.33.6 Data graph properties window displays on the screen..
5.33.7 Select the trace setup.
5.33.8 In data source activate current data, in trace activate the current wavelength, in scale to normalize and in annotation activate the required annotations, and close the window.
5.33.9 Double click on peak report, display the peak report window.
5.33.10 In peak report window activate the current wavelength and require annotations.
5.33.11 Close the method custom report window.
5.33.12 To get the printout of chromatogram, open report, click print and select method custom report and data entered in HPLC logbook.
5.34 After completion of analysis click lamp icon to turn off SPD-M10 A VP lamp.
5.35 Then close the Class-VP window after that close the computer.
5.36 Flush the column with water and methanol for 60minutes each with a flow rate of 1.0 mL / min.
5.37 Disconnect the column from the unit after washing and store in a appropriate place.
5.38 Switch off the pumps and instrument.
Lc solution, acetonitrile, maximum, minimum, acetonitrile, equilibrated
