Chromatography Forum

Dear all,

I don't think I have ever come across a pH gradient when working in reversed-phase or in "Primesep-mode".

I am working with peptides, where often the related compounds differ very little in structure and RP retention (mostly due to that the substance was purified with RP in the last step). A slow pH gradient would separate the molecules acc to pKa instead

Is there a reason why this is a "stay away" area?

pH manipulation is a common means of getting a better separation, but I suspect that gradients aren't popular because of the peak broadening that occurs when you get too close to the pKa of many compounds. Sharp peaks are easier to get if all of a given analyte is either protonated or not.

My idea is that the retention will drastically decrease when I go up in pH and my carboxylic groups will become charged. That should focus the peaks as in standard gradient chromatography. But I don't know, and as always it is better to do the experiment

Gel electrophoresis is not my field of expertise, but isn't it this the way to separate peptides there?

Mattias,

If you really want to exploit the pKa differences of your analytes, you may find the ion exchange technique most rewarding.

Best Regards

pH gradients are not commonly used in RP, but I have used them when the best pH at the start of a run and the end are only a little different. This was for water-soluble vitamins in a messy matrix.

In ion exchange, pH gradients are not nearly as popular as they could be. If you are separating amphoteric ions (amino acids, peptides, proteins) the substances elute in order of their isoelectic points, to a first approximation. The peaks can be quite well formed because of isoelectric focusing effects. There are various buffer systems that will work, citrate being the old classic for cation exchange over pH 3-7.

There have been a few papers by Kaliszan (over the last 3 to 4 years, if I remember correctly) that used pH gradients specifically to determine the pKa of analytes. Kaliszan has also explained the underlying theories in great detail.

It is a functional approach, especially now that packings with a good stability in the alkaline pH range are available. I have personally used the method of pH flips for efficient sample preparation methods.

And finally: no, the peaks do not become wider, if the pH reaches the pK of an analyte, neither at fixed pH nor with pH gradients.