Column Preparation and Equilibration

If your water passes this test at the wavelength you will be using for your chromatography, you are ready to use it to equilibrate the column. The next step is to flush out the dry system and prepare to add the column. 

As soon as the pump begins to pump solvent by itself tighten down the outlet compression fitting and drop the flow rate to about 1 mL/min.The pump is ready to run and should be allowed to pump into a beaker for a few minutes to wash out any machining oils, if new, or soluble residues or dissolved buffer if old. 

Now we can move past the flush valve to the next major system component, the injector. Whichever position you find the injector handle in, leave it 

change the solvent in the reservoir to 70% acetonitrile in water, turn the pump on, and flush the column with the new solvent. Turn the flow rate up to l.OmL/min while catching the column effluent in a beaker. Check the back-up line for leaks if you see any, tighten the appropriate fittings until the leaks just stop. You will always have leaks If you do not, you are probably overtightening your fittings. Leaks are messy, but are probably a sign of successful technique (leaks, not streams). 

Check the pump pressure. The pump pressure gauge and the baseline trace are the two major tools for diagnosing system problems. If the column was shipped in isopropanol or methanol it should start high (3,000-4,000psi) then slowly drop to around 2,000-3,000 psi. 

---