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High Performance Liquid Chromatography Shimadzu LabSolutions Standard Operating Procedure (SOP)

Prepared by: Ziaur Rahaman Asst. Manager (Application)


AQ Chowdhury Science & Synergy (Pvt.) Ltd.
"Plasma Plus+" House #12, Road #5A, Sector #11, Uttara Model Town, Dhaka-1230

TURNING ON THE INSTRUMENT: A. Turn on UPS B. Power on the controller/ Pump A. Power on the modules other than system controller in any sequence C. Turn on computer and Printer

PURGING THE MOBILE PHASE:

Put the mobile phase in the reservoir tray and then:


A. Open the drain valve of the pump module B. Push the purge button C. Purging will start and it will automatically stop after 3 minutes. To stop purging before 3 minutes press the purge button again and close the drain valve. If purging stops automatically only close the drain valve. 1. Double-click the (LabSolutions) icon on the Desktop. 2. Select [Admin] in the [User ID] list for the first login to the system, and click [OK].The [LabSolutions Main] window opens.

3. Click the

icon.

4. Select and double-click the instrument that will be used for data acquisition. The [Realtime Analysis] program opens. A beep sound will be heard if software is successfully connected to the instrument.

Note: If the assistant bar.

(Data Acquisition) icon is not displayed, click

on the title of the

5. Ensure that [Ready] is displayed on the status display in the [Data Acquisition] window. Set the Instrument Parameters: 6. Click [Advanced] to open the [Advanced] sub-window. 7. Click [Data Acquisition] and set end time 8. Click [Pump] Select mode: Binary gradient, set Total flow: 2mL/min, Set B Conc: 50%, Set Maximum Pressure for Pump A & B: 250 kgf/cm2 9. Click [Detector] Set Wavelength CH 1: 325 nm 10. Click [Column Oven] and Set Oven Temperature:40C [ Ambient+15C or 40C] 11. Click [Save Method File As] on the [File] menu. 12. Enter the file name and click [Save]. 13. Click 14. Click the . (Instrument On/Off) button on the toolbar.

Single Run
15. Click the (Start Single Run) icon on the [Acquisition] assistant bar.

16. Set the acquisition conditions, and click [OK]. a. Enter the data file name. b. Select [Auto-increment] and the number type. The data file name is automatically appended with an incremental number.

c. Enter the position of the sample for [Vial#] and [Tray]:1 d. Enter the sample injection volume: 10L Single run is started. Data acquisition ends when the data acquisition time in the method file has elapsed.

Change the End Time during Data Acquisition


17. Click [Change Acquisition Time] on the [Acquisition] menu. 18. Enter an [Acquisition Time], and click [OK].

Stop Single Run


19. Click the (Stop) icon on the [Acquisition] assistant bar. Data acquisition is stopped,

and a data file is created for the data so far.

Realtime Batch
20. Click the (Realtime Batch) icon on the [Main] assistant bar in the [Realtime Analysis]

Program to display the Batch Table.

Enter the sample information, vial #,Tray#, Sample Name, Sample ID, method file name, and data file name and Injection Volume in a Batch Table to sequentially acquire data from multiple samples. a. Click [New] at [Batch Table] to create a new Batch Table file. b. Right Click and the click [Add row] to add rows to the currently displayed Batch Table. c. Enter the sample information, vial #,Tray#, Sample Name, Sample ID, Sample type,method file name, and data file name and Injection Volume in a Batch Table to sequentially acquire data from multiple samples.

d. Save the Batch Table

Data Acquisition Using Batch Tables


21. Select the row numbers to be analyzed. 22. Click the (Start Realtime Batch) icon on the [Realtime Batch] assistant bar. 23. Check [Selected Row(s)], and click [Start]. 24. In this screen, click [OK]. Batch processing of the selected rows is executed.

Stop Realtime Batch


25. Click the (Stop) icon on the [Realtime Batch] assistant bar. 26. Select the processes to stop, and click [OK]. Batch processing is stopped.

Pause Realtime Batch to Edit the Batch Table


27. During realtime batch, click the assistant bar. (Edit Table/Restart) icon on the [Realtime Batch]

28. Set the row where realtime batch is to be paused, and click [OK]. Data acquisition goes into the standby state at the selected row, and batch processing is paused. 29. Select the row to be deleted on the Batch Table, right-click on the selected row, and click [Delete Row]. 30. Click the (Save) button on the toolbar.

31. Click the (Edit Table/Restart) icon on the [Realtime Batch] assistant bar. Batch processing is resumed from the paused row.

Shutdown Analytical Instruments After Data Acquisition


32. Click the (Settings) icon on the [Realtime Batch] assistant bar.

33. Click the [Shutdown] tab, enter the conditions for automatic shutdown of the analytical instruments, and click [OK]. a. Select [Shutdown].

b. Select [Shutdown Method File], and enter the name of the method file that contains the parameters for shutdown of the analytical instruments. 34. Click the (Save) button on the toolbar.

CALCULATION:

1. Click the icon in the [LabSolutions] Main window. 2. Double-click [Postrun]. 3. Click the
4. 5. 6. 7. 8. 9. 10. 11.

(Data Analysis) icon assistant bar to open the [Data Analysis] window.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

By Data Explorer, find out the Data file(Where it was Saved) Open a Standard Data By Double click on it, Click Wizard, A window Will appear Set Width: = 5, Slope=200, Mim Area/ Height= 100000 or other. Click Next Select Box by its Retention time Click Next Set Quantitative Method = External Standard, # of calibration level[If you use one standard concentration give 1(one point calibration), If you use two different standard concentrations give 2(two point calibration),If you use three different standard concentrations give 3(3 point calibration)], unit: ppm Click Next Click Next Type Name:Retinol and set conc. of Std Click FINISH Click APPLY TO METHOD. Click Yes. Click Yes. Save Method As window appears. Give a method file name and Click Save. Click Yes or OK all the times if other things appear. Click Postrun Batch on the Assistant Bar Click File>New Batch File to create batch file for calculation. Click Data Tab on the Data Explorer. Select the one standard data file and drag it to the first row. Right click mouse keeping the mouse on batch Table. Click ADD ROW to add another row in the table. Click Data Tab on the Data Explorer Drag the unknown data file to the last row created Right Click on batch table Click Table Style Select Dilution Factor from the left panel and click Right directed arrow. Enter the values of the item in the batch table as shown below. Click file .Then Save Batch File As and Give a file name. Then Click Save Click Start Postrun Batch. Batch Calculation process will start. If any other windows appear after clicking Start, click OK or Yes.

Dilution Factor, D.F (or Multiplication Factor): For Single Component, 1/VStandard *V1/V2* *V4 Dilution Factor =
1/V Unknown *V1 /V2 **V4

* P* W

Where

W= Average

Weight of Sample (Unknown)

V2 = Second dilution of Standard V2 =Second dilution of Sample V4 = Injection volume

P= Potency =93.5% i.e. 93.5/100 etc.

REPORTS:
REPORT (External Standard): 1. 2. 3. 4. 5. Click Report Formate on Assistant Bar Click File. Then click Open Report Format File and open a report formate. Drag & drop one of the Data file on the Sample Information Table Drag & drop the several Data file on the Summery Compound Table for Standard Drag & drop the several Data file on the Summery Compound Table for Sample.

Print Report:
Click Print Icon and then OK to complete printing.