Chromatography Forum

Dear All,

Being new in LC field I found it's very helpful browsing through the discussions and sharing the experiences.

I'm using perfluorocarboxylic acids as my IPR on my C18 column, should I flush the column everyday after its use? even I'm using it next day? my mobile phase is A: water and IRP, B: ACN. Some papers mentioned that they regenerate the column with ACN, methanol. etc. How often should I do it? And how do I know that the column is well equilibrated? I'm using a capillary column with 4 uL/min.

Any suggestion is appreciated!!

Deling

There is always a trade-off between changes to the column caused by the agents in your mobile phase and the work involved to reequilibrate the column.

My rule of thumb is that I do not recommend to store the column in a specific storage solvent, if the column is used in the same mobile phase every day. If the column is not used over the weekend, I recommend to store the column in a pure organic solvent such as acetonitrile.

These are rules of thumb. If the mobile phase used is an acetate buffer at pH 4.5, you probably would not need to do any conversions to acetonitrile. The stability of a packing in such a mobile phase and in an organic solvent is probably not relevant for practical purposes (i.e. it is like comparing a column life time of 50 years to one of 5000 years).

I like always to flush the column with something like 90%ACN and water, assuming that the re-equilibration of the column will not take more than 5 hours. In general when I would finish with my IPR experiments and I'll have to use the column the next day, before I'll leave the lab I would set up a program in the LC that would flush the column for half an hour with ACN, then for several hours I set up the flow rate would be 0 (for the night), then depending on the time needed for the re-equilibration I would set up in such way that when I'll arrive in the lab the system would be already equilibrated.

Let's say that the column needed 3 hours to be equilibrated, I calculated so that the system will start at 6:00 am with mobile phase A (water and IPR) so when I would arrive in the lab (at 9:00) the column is ready to go...