Chromatography Forum

Hello!

I am having some problems with retention times on a PolyhydroxyethylA column for a ten residue peptide (Hydrophilic). I am using 20mM TEAP in 80% ACN pH3.0 (A) and 20mM TEAP+500mM Naclo4 in 80% ACN pH3.0 (B). The gradient is an increasing concentration of Naclo4 (5%/min).
I see a major peak which i think is my peptide of interest but the retention time of this peak keeps changing with each run. Also my peptide is dissolved in 50%100mM ammonium bicarb pH7.5+ 50% of solvent A. I have the column temp at T=50C during my run.

Can anybody offer any advice on the method or sample preparation or the cause for changing retention times?.

Thanks!

Roy.

Hi Roy,

I can’t really classify the chromatography technique you’re describing but it’s not HILIC nor SEC.
If it were HILIC you should start with a lot of ACN (80-90 % for instance) and run a gradient towards more and more water, potentially with some chaotropic salt in it, but in much lower concentration than 500 mM.

The SEC technique is farther yet from what you’re describing.

What I think it probably is meant to be (without being certain) is HIC. So here is a quick guide to HIC, you can try:

Mobile phase A: 0.02 M Sodium phosphate pH 7 + 0.8 M Sodium Sulfate
Mobile phase B: 0.02 M Sodium phosphate pH 7 + 5 -10 % ACN

Run a very shallow gradient from 0 % B and 0.5 % B per min until your polypeptide elutes.

I think the temperature can be lowered to 35 – 40 degrees C

Good luck

Thanks for your reply, i will try your suggestion.

Roy

In order to work under HIC (hydrophobic interaction chromatography) conditions, you need an hydrophobic column, so the polyhydroxyethyl Aspartamide column won't do the job.

Try to use it under HILIC conditions (i.e. high to low ACN) and constant salt concentration. Try also to match the pH and salt concentration of your mobile phase and injection solvent. If ever you get tailing peaks, then you can also increase the ionic strength of mobile phase B in steps until you get satisfactory results. Be sure that you let enough time for the column to re-equilibrate for reproducible retention times...

Thanks for the suggestion. Will try it.

Hi Roy and Kostas,

My message was actually as follows: A development of a method requires a clear idea of what the added components and the chosen parameters will contribute with. In other words one should find out which technique is most suitable for the method of analysis to be developed and work within it’s limitations, however blurred they are.
In Roy’s situation, I’m not sure HILIC is the right choice. You see, not many - if any - peptides/proteins are soluble in 80 - 90 % ACN so there is a high probability of precipitating the analyte long before it reaches the column (which by the way isn’t very desirable situation either). I would try a number of other options, if I should develop a method for a peptide/protein analysis.
HIC is a fine and “mildâ€