Chromatography Forum

Is it possible to generate a tailing peak via increasing column temperature that isn't a real impurity? In other words can we induce a small level peak split of the main compound, but both compounds revert to the same form at ambient?

Hi Mike H.,

Yes, if the column temperature is high (in my case, 60 degrees Celsius) AND the mobile phase/eluent lines are not preheated. Late eluting peaks both broadened and tailed. Heating the eluent (isocratic method) and insulating the eluent transfer capillary cured this for me.

Is it possible to generate a tailing peak via increasing column temperature that isn't a real impurity? In other words can we induce a small level peak split of the main compound, but both compounds revert to the same form at ambient?

It is also possible that a change in selectivity with temperature pulls a small peak out from under a big one.

Peter

It's actually more common the other way around: tailing/splitting at lower temperature that improves at higher temperature. The "poster child" for this is glucose. A number of columns (notably the heavy-metal form cation exchangers) are capable of separating the alpha- and beta- anomers of glucose. At "room temperature", the anomerization is fairly slow (comparable to the residence time on the column) so you can see a range of bizarre peak shapes (tailing, flat-top, or even split peaks if you go sub-ambient). At higher temperatures (in practice, around 65 degrees C), the anomization is fast enough that you see a single, well-formed peak representing the "average".

Actually, I've seen one example of HIC (Hydrophobic Interaction) of proteins where different conformational states of a protein are more stable at different temperatures (essentially, multi-stage denaturation). Starting at fairly low temperature where one conformer (presumably the native form of the protein) predominated, the peak shape is good. Runs at higher temperature generate tailing peaks as there are two conformers in equilibrium. At still higher temperature, the peak shape improves again as the denatured form predominates.

http://i1194.photobucket.com/albums/aa3 ... 235/40.png

http://i1194.photobucket.com/albums/aa3 ... 235/50.png

This is what the peak splitting looks like. I'm assuming that the ionization state can also change as a result of temperature. Could this also cause potential peak splitting?

Hi Mike H.,

The photos, to my old eyes, seem to show that the largest peak is split and the smaller ones are tailing.

Did you try reversing the column and flushing it out to see if the entry frit is dirty?

Turns out I was causing the peak through method conditions. Compound had a COOH group. I was operating at pH 2.8. I needed to go lower since general Pka of COOH group is ~4, but couldn't go lower because the compound needed to also be buffered to deal with all the N in the molecule and had to be MS friendly. Anyway solution was to use a 50mmol NH4Ac pH 5.8. Chromatography looks good. Tailing is 1.1 now and MS confirms no peak under main peak. Everybody thx for helping me.