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SOP for HPLC System Using Chemstation Software from Agilent

To lay down a procedure for the operation of the HPLC system using Chemstation software.

This is applicable for the operation of the HPLC system using Chemstation software for the Agilent HPLC system.

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Operation of High-performance Liquid Chromatograph
  • Ensure that the HPLC System is placed on a clean and dust-free platform.
  • Ensure that the instrument has been connected to the power supply.
  • Ensure that all communication cords between the units are correctly connected.
  • Switch on the mains and press the power switch of all modules.
  • Prepare the mobile phase as mentioned in the standard test procedure of a specific product. Fill it in the reservoir bottle. Suspend the inlet tube into the reservoir bottle and ensure that the filter is below the level of the mobile phase.
  • Ensure that the appropriate solvent mixture is pumped for seal wash.
  • Ensure that the outlet tube from the detector is dipped into a waste bottle.
  • Connect the column specified in the individual test procedure in the column oven compartment in the flow direction marked on the column.
  • Switch on the mains of the PC. After the completion of self-diagnosis, the main menu of the workstation is displayed.
  • Double-click the Chemstation icon on the desktop screen to open the Chemistation software.

  • Switch on the instrument (Including Injector, Pump, Thermostat, Column and UV lamp) by clicking the “ON” button on the extreme left middle corner of the screen .and screen will be change red to green.
  • Open the purge valve of the instrument and purge the corresponding solvent line(s) in which mobile phase have been placed by setting the flow rate of the pump as 5.0 ml/min, using “Setup Pump” menu from the panel for few minutes until the solvent line is completely filled with new mobile phase.
  • Ensure that no air bubbles are noticed in the solvent line.

Procedure for Method creation
  • Click on the Method folder of respective instrument and create a new method.
  • To create a new method click on the method file menu, click, input a comment for old method from the window displayed and click OK.
  • To create a new method click on method file menu, open “Edit Entire Method” displayed and click OK
  • Enter method information if any and then click OK.
  • In “Set up Injector” option enter the injection volume as per requirement. Injection needle wash option highlighted and input solvent wash vial number and click OK.
  • In “Set up Pump” options input flow rate value per/ml of flow required (i.e. isocratic/ gradient) from “gradient type” create time table program for pump as per test requirement. Input stop time for run sample and set maximum pressure to 400psi. And click OK.
  • In “Column Thermostat Method” option, input the required Temperature value and click OK. Then open signal details option click OK. Open Edit Integration Events click OK.
  • In “VWD Signal” option enter the wave length number by numerical key. If required wave length change during analysis create time table program as per requirement, and then click OK.
  • In “Specific Report” option select the report style e.g. ABC, ABC-1, PERFORMANCE etc. and the click OK.

  • In “Instrument Curve” option select required data curve and the click OK. In “Run Time Checklist ” option select required method section to run and the click OK
  • In the finish window at the end enter a title to describe the program. Open the ‘file’ menu click “save as” option open “method” and input method name with the help of words key and click OK.
  • The software prompts you for a program name to save the program. After giving the program name, ensure that the program is saved under the program folder of the respective instrument.

Procedure for Method single run
  • To open the ‘Method’ file and select the ‘load method’ option open and select required method which is stored in the file and click OK.
  • Now load the select method and its parameter.
  • To open the ‘Run Control’ file and select ‘Sample Information’ option and enter Operator name, Subdirectory name, In data file select manual option and enter data file name. In ‘Sample parameter’ enter vial location number, sample name, sample amount and comment (if any) then click ‘Run Method’.
  • Now run the method and screen change green to dark blue.

Procedure for Sequence creation
  • To create a new sequence click on Sequence file menu, click “New Sequence”. Default sequence will be appear.
  • Open the ‘Sequence” file menu and click ‘sequence table’ enter in “Template for sample name” types a sample name, select Method name and number of vials, injection per vial, and click OK.
  • Open the ‘file’ menu click “save as” option open the “sequence ” and enter sequence name to save the sequence, give a title for sequence, and select a location for data source and appropriate directory to save the sequence as applicable. Click OK.
  • The sequence will be displayed which can be edited as required for sample name. Select the vial position as applicable. The injection volume, programme name, method name, status (of injection), injection time and date are automatically displayed. Save any changes made in the sequence.

Procedure for Sequence run
  • To run a sequence, open the ‘Run Control’ file, click ‘Run Sequence’. The instrument runs automatically after some time.
  • A “Injection List” screen is opened displaying the name of the sequence and comment name on the screen.
  • While the sequence is running, chromatogram can be viewed online from the panel.

Procedure for Data Analysis
  • Single click on the ‘Data Analysis’ folder open the particular file and with sequence (after the run of that sample is completed) to view the raw data. The raw data is processed automatically according to the quantification method specified in the sequence.
  • The raw data can be integrated by clicking on the Editor icon at top of the screen and selecting the “Detection” option at the bottom of the window.
  • Peaks can be labeled from the “Peak Table” option at the bottom of the screen in Editor.
  • Save the changes made in quantification method.
  • Click on the “Printer Layout” icon at top of the screen. The final report is displayed according to the report style selected (given when preparing the method ).
  • Adjust the scale of the chromatogram if required by clicking on “Edit” option and selecting chromatogram properties. The window of the chromatogram properties will have an option for time scale and signal scale which are adjusted as required. Click OK to close this window.
  • Save any changes made in report format. Click on Print icon at the top of the screen to print the chromatogram. To view the next chromatogram click (next) and take print out similarly.
  • Select performance from report to obtain system suitability parameters wherever applicable.

Procedure for System shut down
  • After completion of analysis, wash the column with water and storage solvent and keep it back in the relevant place.
  • Open the panel window. The display shows control menu. Switch off the UV lamp.
  • Set the flow rate to zero. Switch off the motor of pump, column thermostat and sample thermostat.
  • Close the panel and browser window.
  • Switch off the mains of system controller. After self diagnosis is completed switch off the PC.
  • Switch off the mains and depress the power switch of all the modules.
  • Enter the column & instrument usage details in the logbook.

SOP - Standard Operating Procedure
UV - Ultra Violet
PC - Program Controller
HPLC - High Performance Liquid Chromatography


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