Hi everybody

Could somebody please give some advice on the column equilibration time when your mobile phase has THF. My current mobile phase is : MEOH/H2O/THF/HAC 55:34:11:0.4. At present I am equilibrating my column(luna c18) for about 1hr but my peak at about 25min is shifting. During a 24hr run the peak at 25min would "drift" between 24.5min and 25.5min. I have even tried equilibrating my column overnight but this did not help. I also tried using a column heater @ 30C but the "drift" still occured.
Should I perhaps equilibrate my column with 100% THF and then equilibrate with my mobile phase?

Any suggestions would be greatly appreciated.

Assuming you are using an isocratic method, your column would be well equlibrated after an hour. This means the cause is something else, you have already eliminated temperature as a cause so thats not it. Are you using a helium sparge? If you are, realize that the composition of your pre-mixed mobil phase will change over time as components evaporate out of the bottle. A very small change in mobil phase composition can lead to relativly large changes in retention times. On the other side of the coin, outgassing of your mobil phase can also cause variable RT times by slightly reducing flow rates.

Equilibration with 100% THF then MP will not help your drift, but will remove highly bound components.

I'm not using helium sparging but I'm using an Aliance 2690 which has an in-line degasser. I've taken all the necessary precautions i.e priming all lines, making sure that all lines are in solvent, but I still get the "drift". I've set the degasser on continous but this problem still persisted. I've even performed a PQ on my system obtaining %rsd of less than 0.1% on ret time. Therefore I can safely say the system is not at fault but the there could be some chemistry problems.

A question. Is the peak at 25 min the only one to drift, or do all the peaks in the chromatogram show this drift?

All the peaks are drifting slightly but the peak at 25min is drifting significantly i.e %RSD on ret time of peaks before 10min is less than 1% but %RSD on ret time of peak at 25min is about 3%.

It might be time to check your pump. How stable is the flow rate?

Be careful when using THF in an HPLC: Some fitting and tubing materials (e.g. PEEK) cannot stand up to it.

The composition of my mobile phase is:
A) 0,005 M Ammonium acetate (#): THF 95:5% v/v
B) 0,005 M Ammonium acetate (#): THF 5:95% v/v
How can I wash my column (Zorbax SB18; 5 µm; 4,6 x 250 mm)?
Usually I use 10 column volume of water (#): THF 95%:5% v/v, then I elute the strongly retained compounds with methanol.
Is this procedure correct?

Are you sure there are no strong-retained compounds in your samples? Using an isocratic method you may introduce some compounds with each injection that can not be washed out with your mobile phase. They are just sitting on the column, blocking it and reducing the surface avalable for every next injection. I would try increase percentage of organic component in the mobile phase and see if any peaks would come out. You may even need to try a stronger organic solvent. If the strong-retained compounds are the case, you may need to add a washing cycle to your method. If the retention time consistancy is not very critical, you may just wash the column from time to time. A guard column may help as well.

Now, talking about the column washing (and this should answer Barbieri's question also). This is what HP and Zorbax recommend for their columns:
Flush with stronger solvents than your mobile phase. Following is the list of reversed-phase solvent choices in order of increasing strength (at least 25mL of each solvent should be used for analytical columns):
- mobile phase without buffer salts;
- 100% methanol;
- 100% acetonitrile;
- 75% acetonitrile: 25% isopropanol;
- 100% isopropanol;
- 100% methylene chloride;
- 100% hexane.
When using either methylene chloride or hexane the column should be flushed with isopropanol before returning to your reversed-phase mobile phase.

Know your sample and use common sense when determining what solvents to use and how intensive column washing is needed.

Good luck