SOP for Agilent 1100 HPLC ChemStation

PURPOSE

To lay down the procedure for the operation of Agilent 1100 HPLC with ChemStation.

SCOPE
This SOP is applicable to the Quality control / Stability department.

RESPONSIBILITY
Assistant – QC / Stability to operate the HPLC.

ACCOUNTABILITY
Manager - Quality control / Stability To ensure the compliance for established procedure

PROCEDURE

• Turn on all the modules for the HPLC system (CapPump G1376A, DAD G1315B) and allow for the system to initialize.
• Turn on the computer, and open the ChemStation software by clicking Start --->Programs ---> Agilent Chemstation ---> Instrument 1 online.
• Choose the configuration that will be used (Capillary pump G1376A and DAD G1315B) and click OK.

• Under the “Instrument” menu, choose “System On” to turn on the pump and DAD or use the shortcut button on the instrument control page.

• It is recommended to flush the reservoir lines before your column is connected to the HPLC system. Click at the pump interface ---> Purge… ---> More>>

ALSO READ: SOP for Operation of Agilent 1260 HPLC

• Choose the mobile phase reservoirs that you will use. Purging the reservoir one by one.

• To connect your column to the instrument, turn off the pump and all connections should be finger-tight (do not over-tighten)

ALSO READ: SOP for HPLC System Using Chemstation Software from Agilent

• Turn the pump on and monitor your pressure and ensure that your pressure is within your column’s specifications. Watch for pressure drops which may indicate a leakage problem.

• Set up a method by either starting a New Method or Edit Entire Method. Under “Method” menu, you can choose “New Method” or “Edit Entire Method…”

👉 Click method sections to edit

👉 Choose pump mode to use (Micro Flow / Normal Flow), set the flow rate as well as compositions of mobile phase to use. This setting is isocratic elution.

👉 If gradient elution will be used, set the timetable of the mobile phase.

👉Set the DAD wavelength to monitor (5 channels are available simultaneously.)

👉 Add available signals to the method

👉  Edit integration events for chromatogram

👉 Set Specifications for the report and instrument curve.

👉The instrument data curve (i.e. mobile phase compositions, Flow rate, Temperature, Pressure or UV lamp voltage) can be overlaid to the chromatogram.

👉 After the Method is set, save the method. Under the “File” menu ---> “Save as” ---> “Method”

👉 Put the method name

Run a single sample

• Put the sample information; Under “Run Control” ---> “Sample Info…”

Insert sample information i.e. Operator name, Subdirectory, the File name (prefix/counter can be used.), Sample name, and Comments (The running condition can be entered in this field such as, sample information, mobile phase composition, Flow rate, and Injection volume)

• Start running by clicking “Run Method”

• Let the column to be equilibrated until the smooth baseline is obtained.
• inject the sample by using an HPLC syringe into the injection valve and then switch the injection valve to the “inject” position. The running will start and elapsed time begins.

• If you want to stop the run, Under the “Run Control” Menu, click “Stop Run/Inject/Sequence” or shortcut F8.

Data Analysis

• The chromatogram is processed in the “Data Analysis” Tab (second tab on the left panel).

Load the chromatogram by click “File” ---> “Load Signal” ---> Choose the desired file name

The peaks on the chromatogram can be manually integrated or deleted by using the “integration toolbar”.

• The chromatogram can be printed out

Under File menu ---> Print Preview ---> Report and Click Print

• Once the running is finished, Flush the analytical column with a high percent of methanol or acetonitrile and keep it in a storage solution (see the column manual).
• Turn off the pump and the lamp by clicking the DAD ---> “Control” and click off for both UV and Vis lamps. You can close out the software, and turn off the instrument (Micropump and DAD).

ANNEXURE

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REVISION HISTORY

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ALSO READ: SOP for System Suitability Test in HPLC Analysis and HPLC Documentation