How to recover C18 column after prolonged exposure to phosphate buffer?

Question

A colleague of mine who occasionally does HPLC in a biochemistry lab discovered that a C18 column was not properly flushed after the previous experiment. It appears that the column was stored for several weeks capped with aqueous phosphate buffer inside.

The main concern is partially crystallized phosphate as well as bacterial growth as there is no clear evidence whether antibiotics were added to the buffer or not.

Is there a way to recover the column, and if so, what procedure is typically recommended (solvents, temperature, flow rate etc., preferably with a reference for the lab report)?

Answer 1

The bad news is that HPLC columns often die because this ill-treatment. We have had the same issues and eventually several expensive columns was discarded due to the negligence of a student. Acetonitrile, when used above 90% with a phosphate buffer, leads to precipitation inside the column. I have great deal of experience with column manufacturing technology. You never add antibiotics to mobile phases. So don't worry about that. The solvents themselves, like ACN, and MeOH ensure that bacteria don't survive inside the column. This is the typical storage solvent after washing the column and ensuring all the buffer is out.

The salvaging procedure can be the following:

1. Run the column at really low flow rates say, 0.1 mL/min with a high aqueous content (90/10 H2O/ACN). If the pressure is extensively high change the flow direction. Columns have a flow direction marked on them. Try the direction which shows a lower pressure.

2. Record the pressure, if the pressure slowly goes down, this is a very good news.

3. If the step 2 is going well, wash overnight at 0.1 mL/min. If all went well, pressure is back to normal in the morning, you are good to go.

4. Never sonicate the column. It will damage the bed permanently.

P.S. Phase collapse has nothing to do with problem of precipitation inside the column. This is a completely different story. Please don't use that suggestion from LC-GC magazine (link in the comment above). Ron Majors is very experienced but here he talks about another issue.

Phase collapse is not a well understood phenomenon, a better word is de-wetting. Since the C18 phase is very hydrophobic, with highly aqueous mobile phases, the pores (where all the retention occurs) exclude water. Chromatographers, especially those who write in commercial magazines for public readership, tend to do a lot of imaginations.

If the column pressure becomes normal, repeat and old separation. If the retention time is similar, no need to worry. If it is reduced, then try the LCGC suggestion. Pressure is the main test to confirm if all ppt is gone or not.