Fong:HPLC

UNDER CONSTRUCTION

Overview

This protocol describes the basic use of the Dionex HPLC located in the Fong Lab. If you have never used it before please discuss your experiment plan with Dr. Fong and get someone to show you the following steps. Improper use can result in very costly damage to the HPLC or to the column, both of which are worth more than your pitiful life, you worthless piece of garbage :)

Current Setup<br\> Dionex HPLC <br\> UV variable wavelength detector<br\> Refractive index detector<br\> BioRad column <br\> <br\> 0.050 mM H₂SO₄ eluant<br\> 0.6 mL/min flowrate<br\> 20uL injection volume<br\>

Startup

Use the following steps to start up the HPLC from scratch (this takes about an hour, plan ahead!):

1. Turn on all components
2. Ensure Chromeleon is connected to the HPLC hub by doubleclicking on the icon in the desktop tray. The dialog should say that the server is running idle.
3. Open Chromeleon software package, located on the desktop.
4. Navigate to the Control Panel, either in the Window menu or by selecting the Control Panel in the Browser window.
5. On the home tab, click Connect All, which connects to all components except for the RI detector.
6. On the RI tab, click Connect.
7. If the HPLC has not been used in the last 6 hours or so, purge the pump:
   1. Loosen the pressure release valve on the pump.
   2. On the Pump tab, click Purge On then OK. This will deliver a flow rate of 6.0 mL / min for 5 min to the pump only to purge any air bubbles in the line.
   3. Close the pressure release valve on the pump.
8. On the Pump tab, ensure the flow rate is set to 0.6 mL/min then click Motor On.
9. On the RI tab, set Purge to On, then hit Enter. This purges the RI reference cell.
10. On the Home tab, click Monitor Baseline 'On' and watch for the signal traces on the Home tab. If both traces do not appear, you can add traces by double-clicking the window.
11. Allow the baselines for UV and RI to stabilize before running samples. What constitutes a "stable" baseline is a matter of personal preference, but, generally, there should be no drift greater than ~5-10 units / 30 minutes and no individual peaks larger than ~1. Under normal circumstances, the baselines usually stabilize in about 30-45 minutes.