Chromatography Forum

Hm, there are all sorts of things that might lead to retention time drifting.
So far we can only eliminate some things:
- at 23% organic you definitely won't have a problem with phase dewetting
- a pH of 2.5 - 2.8 should be no problem for a C18 column concerning bonded phase stripping. If it's indeed a XBridge, I'm sure that it's no problem. I've used an XBridge C18 at pH 1.5, 45°C, thousands of injections with no retention loss.
- the fact that fresh mobile phase doesn't change anything rules out the mobile phase changing over time.
Did you try to wash the column with high organic (or even 100% methanol) to see if the initial retention time can be restored? Be sure to thourougly equillibrate your column with that ion-pairing mobile phase after the washing.

Could it be that actually your samples are fouling the column? There is a way of verifying if the mobile phase or the samples are changing the column. Basically, you keep the mobile phase flowing and inject samples at erratic time points. If you see a correlation between the changing parameter (in your case retention time) and number of injections, the samples are the culprit. If, on the other hand, there's a correlation between analyte retention time and total time (i.e. volume of mobile phase flown through the column) then it's a mobile phase incompatibility.

Hi crillyr,

I thought I posted successfully, but I guess something must have happened. So as not to tailgate HPLCaddict too much, I agree with his assessment and the nature of your troubleshooting that has been done.

Checking to see if the samples' matrix is fouling the column is an excellent idea, and HPLCaddict's suggestion is apt. Do injections of standard material in the absence of matrix suffer from a similar loss in retention?

Another thought, and one reason that I hate and try to avoid ion-pairing agents, is that at times a column may take up to a day (though this was for dodecylsulfonate and not octanesulfonate) to properly equilibrate, to become "saturated" with the ion-pairing agent. How long do you typically allow for equilibration of the eluent with the column, and how do you handle the column between sample runs? Probably the better course of action as far as minimizing column equilibration time from run-to-run is to store the column in the eluent if the column is used frequently for running the method, say multiple times per week.

I am curious...what is the range of k (retention factor) for the analyte peaks as they suffer this shift in retention of 20 minutes?

Could it be that actually your samples are fouling the column? There is a way of verifying if the mobile phase or the samples are changing the column. Basically, you keep the mobile phase flowing and inject samples at erratic time points. If you see a correlation between the changing parameter (in your case retention time) and number of injections, the samples are the culprit. If, on the other hand, there's a correlation between analyte retention time and total time (i.e. volume of mobile phase flown through the column) then it's a mobile phase incompatibility.

This is a really great idea! May have to use this if I ever run into such an issue.