Try using a guard column during analysis

Hi, Dear Chromatographers:

I have a question regarding column washing/storage after use each time, specifically, we are analysing basic compounds using RP C18 column, mobile phase - K2HPO4 buffer with TEA/ACN, at the end of run we wash the column with gradient of H2O/ACN and store at 50/50. But we are experiencing repeatability problem even after long time column equlibrating. Is this related to TEA? Could someone suggest a better washing method? Also generally what is the better way to wash a column after using buffer/ion-pair mobile phase?

Thanks in advance.

Dear sir after finsihing your analysis, first you wash with water, then H2O/ACN 50/50 and finally store it in H2O/ACN 20/80

I suggest to wash the column first for one or two hours with H2O/ACN 80:20. If TEA was used or you're afraid that basic compounds were not purged from the column, add acid (no buffer!) to gain a pH around 3 to solve TEA (or bases) from the surface. Stop the flow for some time (30 min or so) and wash again with this eluent for one or two hours. Or programm a method which stops the flow after 30 min. for 10 min, repeating this procedure 5 times.
Then proceed with step 2 and 3 as Anon above described.

If you want to keep your column stable, long term storage (more than three days)should be done in 100% organic, best acetonitrile. For day-to-day storage, it matters little what you do. I would do nothing, to save time the next morning.

Thanks Uwe @ Anons for your suggestions.

When we use Buffers(K-PO4), we normaly wash with H2O - H2O/ACN - ACN, and store in H2O/ACN. It works fine. The problem comes when we use both buffer and ion-pair or modifier like TEA. On several ocassions, after we wash/store the columns the same way, we couldn't get the repeatable results, e.g., RT, peak shape even on the same system and after long time equilibrating. I guess buffer can be easily washed out from the column, but how about ion-pair/TEA? Since IP/TEA modify silica surface, is it neccessary to wash them out every time and then take long time to re-equilibarate/modify the column again when use next time? Maybe do nothing is the way as suggested above?
Thanks again.

With true ion-pair reagents (like octylsulfonic acid, SDS, or a tetrabutyl ammonium salt), it always takes a long time to reequilibrate. The reason is that you need to build up the reagent concentration on the surface again after you have removed it. Therefore I prefer to store a column that is used with an ion-pair reagent in the mobile phase without washing. TEA should not be a strong enough pairing reagent to cause problems. In addition, the molar concentration used with TEA is usually higher than what is used in typical ion-pairing separations. Therefore, for a column that is just used with a TEA buffer, I would go the other route and store the column for long-term storage in an organic solvent.
To store a column in an aqueous system does not improve the lifetime (unless you are using aggressive mobile phases), and it adds to the work load for reequilibration.