How to operate HPLC (High Performance Liquid Chromatography)

1. Turn on the switches of the pump and detector.

2. Turn on the computer first and then printer.

3. The word " Hewlett Packard System Hardware Test" will be shown on monitor and that means the computer is checking the system for examples; harddisk. If the computer does not find any problems, the monitor will show the word "Pass".

4. Next, type " C:\> win " to get into the HPLC system.

5. The word "HP Chemistation" will appears on the monitor and then double click at "Instrument online HPLC system".

6. The monitor will show the word "HPLC system" which gives you "peak" (when the sample is injected), the flowrate of mobile phase or solvent, wave length and pressure.

7. For the pump, click the button "pump on" and then "enter".

8. For the detector, click the button "lamp on" and then "enter". After that, adjust the magnitude of wavelength as you prefer.

9. Adjust the condition of the pump by keying in the values to the computer at the icon "the Instrument" and "Set up pump".

9.1 Adjust the flowrate of mobile phase starting from 0.1 ml/min

9.2 Select the proper kind of solvent to be the mobile phase; for examples

A. Acetonitrile

B. Water

C: Acetic acid

D. Methanol

The mobile phase can be mixed together, for example B 10%, D 90%

10. Gradually increase the flowrate of the mobile phase by the step of 0.1 ml/min. As the time passes, the pressure increases.When the word "Ready" appears on the monitor, the pressure is constant. Then increase the flowrate of mobile phase one more step. Repeat the same way until reaching the prefered flowrate. Wait until the base line steady then prepare to inject the sample.

11. Clean the syringe with the sample before injecting the sample into HPLC. Then inject and load the sample into HPLC, the components of the sample will be detected by the detector and the time is counted.

12. Peak of each component will be shown on the monitor at the different retention time. When there are no more peaks detected, press F8 button to stop detecting the sample.

13. Computer will calculate and integrate the area of each peak. Then the results will be presented in the relation between area and the retention time. Peaks and other results can be viewed by clicking at the following icons; "View", "Data analysis", "Load signal" and "Integration result".