Chromatography Forum

Having repeatability problems using a gradient. The mobile phase is high aqueous (~5%) moving to high organic (95%) over 20 minutes. After use the columns are flushed and stored in 50/50 water/organic. When the columns are used after storage how should they be re-equilibrated? Our analyst like to condition the column overnight at 0.1ml/min using the starting mobile phase. Can this be causing phase collapse? Should the columns be conditioned using high organic prior to conditioning with the mobile phase?

Thanks

"phase collapse" has been pretty much been deprecated as an explanation for the issues encountered with C18 columns (especially) in high-aqueous mobile phases; "dewetting" is a better explanation (viewtopic.php?f=1&t=6797).

That said, I would not expect dewetting to be an issue on your C8 column with 5% organic in your mobile phase. Unless you have something exotic in your buffer (like ion-pair reagents), you should just be able to flush the column out with your initial mobile phase (about 10 column volumes is usually safe) at the regular flow rate.

The PLRP is a different story. Being polystyrene, it somewhat more likely to shrink/swell with changes in mobile phase composition. Which means that literal phase collapse can occur if the resin swells enough to spike the pressure. 10 column volumes is still probably enough to get washout, but "low and slow" is probably a good idea. 0.1 mL/min overnight may be overkill; what you could do is to try it at a higher flow and watch the pressure to see what happens.

Having repeatability problems using a gradient. The mobile phase is high aqueous (~5%) moving to high organic (95%) over 20 minutes. After use the columns are flushed and stored in 50/50 water/organic. When the columns are used after storage how should they be re-equilibrated? Our analyst like to condition the column overnight at 0.1ml/min using the starting mobile phase. Can this be causing phase collapse? Should the columns be conditioned using high organic prior to conditioning with the mobile phase?

Thanks

There is no bonded phase on PLRP-S since those are polymeric phases. I am not sure about C8 phase collapse ( whether the alkyl chains are flexible enough?). Your gradient is very steep and polymeric phases have some tendency to swell to some extent (it is minimum when the cross-link is high). Have you tried increasing equilibration time between the runs?