Recently, I have been getting continuously increasing retention times on the following ion pairing method:

150x4.6mmx5um C8 column. Flow rate of 1.6mL/min with 38/62 MeOH/50mM Na Acetate pH 5.0 + 15mM SLS. Column at 40C. HP 1100 system (but Waters autosampler) with UV Detector. 20uL injections. Sample diluent is matched to the mobile phase (minus the IP agent).

There is no change in void. I have 5 drug/degradant peaks which are cations at this pH that are increasing in retention by as much as 10% overnight during repeated injections. One excipient (possibly neutral aromatic) loses retention at the same time and fails to separate from a drug peak. Pre-mixed or MCGV-mixed get me the same thing.

The confusing thing is I was not seeing this 2 months ago...

Columns sometimes take incredibly long to equilibrate with ion-pair reagents. I've heard as long as a couple of days. My guess would be an equilibration problem and that as soon as equilibrium is reached, your RT's will stabilize.

is retention always increasing? You state that retention increased overnight. Had that problem once and it turned out the temperature in the lab was plumeting during the night, and thereby increasing time. Or, you might be loosing methanol some way ( is the eluent premixed) You might occasionally inject something that does not ion pair and see if its retention also increases. If it doesn't then the anonymous's suggestion is your problem.

Thanks for the input. I set that system up to run over this past weekend and now it looks like it may have finally equilibrated. Three days?! Our QC lab is going to be throwing darts at pictures of my group.

Does anyone have any tips for accelerating equilibration on an IP system? Is it worthwhile to increase the flow rate for the first several hours?

I have seen this problem with SLS before depending on the manufacturer/purity.

I think there could be a very low percentage impurity/impurities in SLS that is more strongly adsorbed to the stationary phase than SLS.

For example, if there is a very small amount of C-13 sulfate then the column would first equilibrate with SLS then since C-13 would be more strongly retained it would slowly displace the C-12 sulfate and since it is more strongly retained it would reach a higher adsorbed concentration and the net charge on the column would slowly increase and retention times could slowly increase.

Anyway, just my speculation. I have seen this with SLS before and it is never my first choice in ion paring reagent.

I also had a problem like this some years ago. The RT seemed to be stable, but also increased overnight. it drove us crazy. We tried all kinds of things. Finally, we too, found that it was temperature related (it gets cold at night in Feb in NJ and the thermostats were turned down). When we used a column heater, the problem miraculously disappeared. I would try running all day and see if the RT increases and then try running at night when the temp goes down.

We had heard of temp effects during IP methods so we've been running in a heater at 40C the entire time.

The possibility of a highly non-polar impurity was brought up here and may very well be part of the problem. Unfortunately when we tried using a high purity SLS (Fisher's 99%), instead of the JT Baker 95% material we are using, the separation was poor. It appears that some of our peaks like the JT Baker impurities!

For the moment we are letting the columns equilibrate for the apparently requisite 2 days.

Some excellent insights gentlemen. I appreciate your counsel.