PKKK/300/UP/044 HPLC Agilent 1

General Operation of HPLC System H

(For Identification and Quantitation Using SPD-M20A Diode Array Detector and Low Pressure Gradient HPLC)

Power on each Agilent HPLC (binary pump, vacuum degasser, auto sampler, column compartment, diode array detector).

From the Start Menu, select All Programs, Agilent Chemstation, and Instrument 1 Online. Or double click the Instrument 1 on the desktop.

Install required column. Prepare the mobile phases A and B and place the respective suction filter line into the reservoirs.

After purging, turn off the pump. Close the purge valve.

Creating the method.

From the ‘Method’ menu, select ‘Edit Entire Method’.

Make sure the UV and Visible lamps are turn on and select the prerun autobalance option is active.

If running cosmetics samples for identification of tretinoin and hydroquinone, select acquisition of all spectra using the full spectral range of 190-950 nm and peak width that corresponds to one spectra every 0.1 seconds.

Tick the ‘Data Acquisition’ and ‘Standard Data Analysis’ to execute at the run time.

From the ‘Method’ menu, select ‘Save Method As’, key in a filename. OK

To inject an individual sample, click ‘Run Control’, select ‘Sample Info’ and fill in the “Operator Name”, ‘Subdirectory”, “Location of the Vial”, “Sample Name” and “Injection Volume”

Under Sequence menu, select Sequence Parameters. Create Subdirectory. Select Auto for the data file naming system.

Under Sequence menu, select New Sequence. Fill in the Sequence Table and Save.

Data Analysis (Integration)

To Auto Integrate, click “Integration Menu” and then click “Auto Integrate”. Auto Integration will display the defaults event.

To name the peaks, select the “Calibration Menu”, choose “New Calibration Table”, click “Ok”. Key in the compound name and then click “Ok”. Click “No” for “Delete lines with zero amounts?”.

Choose “Graphics Menu” and select “Signal Option”. Tick “Axes”, “Baseline”, “Tick Marks”, “Compounds Name” and “Retention Times”. Click “Ok”. Signal option is used to define a look of chromatogram when it is produced on the screen or the printer.

To print the result.

Change Channel A from mobile phase to 5% Methanol and Channel B to 100% Methanol.

After purging, turn off the pump. Close the purge valve.

Shutdown the system by selecting “Instrument Menu” and click “System Off”. Power on each Agilent HPLC (binary pump, vacuum degasser, auto sampler, column compartment, diode array detector).

Precautions

Performance Check will be carried out in every analysis to ensure the sensitivity for the instrument is always at its optimum level. It is carried out by performing the System Suitability Test using the Standard solution/ Mix Standard Solution and to ensure that the parameters below are fulfilled:

Tailing factor < 2.0

Theoretical plates > 1500