Chromatography Forum

I made a method that separates 14 compounds, but I am having a problem with one of the compounds and I hope someone can give me ideas about how to approach it. The problem is that one of the imps is symmetrically broad with low plates where all the others are sharp with high plates. All compounds have a similar backbone with -COOH, amides and -OH substituents, but the broad peak has an aldehyde group and since this is the only difference from other compounds that have sharp peaks, it must be the cause.

Can someone list some potential cause for symmetrical broad peaks.

The gradient is straight forward 10mmol PPM, pH 2.5, ACN, 10% MeOH (constant. This is in method because I had 2 alcohol imps that fronted a lot early in the chromatogram. The inclusion improved the asymmetry from 0.7 to 1.1). Column is SB-Aq phase 100mm, 4.6mm x 1.8µm.

Any suggestions are welcomed. thx

Interesting problem.
Is it possible that the aldehyde group leads to some sort of keto-enol-tautomerism? This should usually be pretty fast and not observable in the timescale of HPLC, but if the isomerism is somewhat hindered...I'd expect some sort of "saddle" peak if isomerism is the case, but maybe it leads to broadening here?? Or the aldehyde group might cause some sort of ring structure via H-Bonding with an alcohole?

Did you try higher column temperature? If hindered isomerism is the culprit, higher temperature should speed up the process and therefore sharpen the peak. Might kill your selectivity elsewhere, of course.

I will try a higher temp. Also looking at the structure there is a -COOH that might be close enough to potentially interact with aldehyde, if this is a possibility. thx

Another possibility might be that the MP's pH is close to the pKa of that particular analyte. A pH change to the MP might fix it (or cause similar issues with a different peak).

Maybe the use of annother chromatographic mode can help, HILIC for example. And you can play around with column lengh and a smaller ID of the column.

Try to remove methanol from the mobile phase. May be you are forming acetals and the equilibrium is affecting your peak shape. Also if the column has residual amino around (from hydrolysis of polar embedded stationary phase) it might form imine.

Thanks all. Turns out that the equilibrium keto enol can be pushed to one state via pH. New method resolves all compounds, except two metabolite compounds which are not critical in final product since they occur in the body. Conditions are pH 10.0 NH4Fm with 1.4g EDTA/L.

Thanks all. Turns out that the equilibrium keto enol can be pushed to one state via pH. New method resolves all compounds, except two metabolite compounds which are not critical in final product since they occur in the body. Conditions are pH 10.0 NH4Fm with 1.4g EDTA/L.

::fist pump::