Chromatography Forum

Does concentration of buffer of mobile phase have an effect on retention time in reversed phase chromatography,for eg; If I change my buffer concentration from 0.3 mM to 0.6 mM ,will it have any effect on retention time for the peak of interest in RPC?

Most of the time, so long as there is sufficient buffer to control ionization, the actual concentration above that has little effect on retention. Typical buffer concentrations for LC with UV detection are in the 10 - 50 mM range. At below 1 mM, you may be below the "suficient" level.

Does concentration of buffer of mobile phase have an effect on retention time in reversed phase chromatography,for eg; If I change my buffer concentration from 0.3 mM to 0.6 mM ,will it have any effect on retention time for the peak of interest in RPC?

ESK - you're the researcher - research this !!! This is the type of thing done in robustness studies, like how far can the buffer concentration vary without affecting the results !!!

I did a study on this with some basic drugs since the silica plays a role in retention. At 2.5 mM buffer concentration k' was over 10, at 50 mM k' was 4. Tailing inmproved significantly as well.

The pH was the same?

Yes, it was a low pH phosphate buffer.

I did a study on this with some basic drugs since the silica plays a role in retention. At 2.5 mM buffer concentration k' was over 10, at 50 mM k' was 4. Tailing improved significantly as well

I'm not surprised, but it would have been interesting to see intermediate results. Based on my experience, I would expect to see a big improvement from 2.5 to 5 to 10 and then leveling off. The results would depend quite a bit on the on the silanol activity as well. Newer ("type B", "high purity", . . . etc.) packings can tolerate lower buffer concentrations. Regardless, if the concentration is below 1 mM, I expect that it *will* matter.

What is "low pH"? I am still much surprised at such a large ionic strength effect if the pH was low enough to eliminate SiO-.

pH was 2.5, done on an older silica column. Yes, both the tailing and the effect on k leveled off. Not much improvement above 20 mM for the compounds I tested.

Do you have organic modifyer in your mobile phase? How did you determine pH? I still think this is a pH effect, if it is really an ionic strength effect I would be strongly interested of the identiy of your compounds.

pH might be low enough in principle, but if the buffer concentration is too low (hence low buffer capacity) the residual silanols will still cause trouble – especially (but not exclusively) in the case of the silica A support.

Best Regards

Monitoring the effect of buffer concentration on retention and peak shape is the classic experiment to determine the extent to which ion exchange plays a role in the separation.

Tom's advice is sound regarding the typical minimum buffer concentration (5-10mM) to mask the ion exchange effects that will be observable with basic compounds on many of the newest packings out there.