Chromatography Forum

Hello,
I am analysed a peptide with an important number of Lysines by LC-MS. To increase the retention on the column (CSH) I am working with HFBA. A gradient with mobile phase: HFBA (0.1%) in water /HFBA (0.1%) in acetontrile. The retention is Ok and the sensitivity as well, but the columns (CSH) have a short life. I think the additive is the cause of the problem, but I am not sure. Has anybody seen this problem before?
Any idea? I am thinking of adding ammonium formate to increase the pH.
Thanks a lot for your time!

HFBA if injected will react to form heptafluorobutyric acid which is a fairly strong acid and will attack the column. In GC it is necessary to remove it by doing a water/organic extraction or to evaporate the sample and redissolve it. I believe I read using alkaline will degrade the derivatized compounds, you should use pH 6 phosphate buffer or similar.

Lysine is a very hydrophilic amino acid. Why not analyze your peptide using HILIC? Specifically, use an anion-exchange column in the HILIC mode: Start with 85 -90% ACN containing 10 mM ammonium acetate (overall concentration) and run a gradient to 20% ACN containing something like 0.2% formic acid. The peptide will experience electrostatic repulsion from the stationary phase but the high initial concentration of ACN will superimpose hydrophilic interaction strong enough to force the peptide to be retained anyway. This combination is called ERLIC (Electrostatic Repulsion-Hydrophilic Interaction Chromatography). As one decreases the ACN concentration, one tunes down the hydrophilic interaction to the point that the peptide is then eluted by the repulsion effects.

Thanks a lot for your comments

HFBA if injected will react to form heptafluorobutyric acid which is a fairly strong acid and will attack the column. In GC it is necessary to remove it by doing a water/organic extraction or to evaporate the sample and redissolve it. I believe I read using alkaline will degrade the derivatized compounds, you should use pH 6 phosphate buffer or similar.

She/he is using an acid not anhydride. You never inject anhydride into aqueous mobile phases, you need to quench it first.

Hello,
I am analysed a peptide with an important number of Lysines by LC-MS. To increase the retention on the column (CSH) I am working with HFBA. A gradient with mobile phase: HFBA (0.1%) in water /HFBA (0.1%) in acetontrile. The retention is Ok and the sensitivity as well, but the columns (CSH) have a short life. I think the additive is the cause of the problem, but I am not sure. Has anybody seen this problem before?
Any idea? I am thinking of adding ammonium formate to increase the pH.
Thanks a lot for your time!

Depending on the size of your peptide and number of basic groups you might be able to use mixed-mode column with some ACN/water/ammonium formate mobile phase, but the approach Andrew offered should work too. The only obstacle you might have is solubility of your peptide in high organic. If you sample is in water you want to avoid injecting high volume injection into high concentration of ACN to avoid peak disturbance. Since you will be exploring ion-exclusion and HILIC, more buffer/ions will mean more retention for peptide and less retention for your acidic counterions (if you have them)

HFBA can also contaminate and damage many types of vacuum degasser modules too, resulting in contamination of the entire flow path, detector, column and so on. Stay away from it.

Thanks a lot for your comments.
Alpert I have read you papers about this. it is very interesting.
Thanks