Chromatography Forum

Hi,

Could complexation of phosphates with metal parts of the HPLC system cause peak tailing ?

Does this complexation occur also under basic conditions ? (20 mM ammonium acetate pH 10)

If yes, how can I remove the adsorbed phosphates ? (could EDTA help?)

Any suggestions? Thanks.

Maybe 2 things to try:

- Different buffer (ammonium hydrogen carbonate)
- Increase buffer concentration (50mM)

Maybe also pH 10 TEAA buffer (to ion-pair with the phosphate group)

Only if the metal ( usually Stainless Steel ) surfaces were active, which should not occur during normal use.

I assume you are talking about phosphate buffers, and affect on other analytes, not trying to analyse phosphate by IC using a stainless steel instrument, which does have problems due to high pH mobile phase creating activity and other problems.

The solution would be to passivate the metallic parts of the system using manufacturer's protocols. Obviously, remove fragile components, such as column, guard column.

The most common passivating agent for SS has been nitric acid, but citric acid passivation is increasingly popular and can be as effective, and also offers environmental and safety benefits if you unfortunately have fascists posing as chemical safety officers.

Please keep having fun

Bruce Hamilton

if you unfortunately have fascists posing as chemical safety officers

Does it apply to having clowns as well? In addition to the passivation instructions above, disconnect the detector during the initial flush. I did it on a system once where a dark rust colored liquid came out (with 10% HNO3) and the system started to shoot out leaks. It wasn't for ion analysis, but it was truly a system held together by rust. It was an old system dedicated for radioactive analysis, almost exclusively running a gradient on a microbondapak column into a scintillation counter. How many things wrong can you find in this post?

Hi,
I have no problems with metal HPLC systems and phosphopeptides. And I do not use any basic buffers for separation of phosphorylated peptides, just "normal" acetonitrile/water gradient. Peak tailing of phosphopeptides on C18 phases is, unfortunately, a reality. Of course, you could go on and try to introduce different ion-pairing agents but it will not help to much. The reason for peak tailing of multiple phosphorylated phosphopeptides is that you have increased hydrophobicity with increasing number of phospho groups (more negative phospho groups will neutralize the positive charge on amino groups and your peptide will get more and more neutral and hydrophobic). Try to use some base deactivated C18 phase, like Waters BEH C18 or Luna from Phenomenex. Some people say that the sample should be dissolved in 50 mM citric acid and that it will bind all of free metal ions in mobile phase and on the column surface. However, I did test the approach with 5 different stationary phases and could not confirm the finding.
Success

I disagree that increasing number of phospho groups will increase the hydrophobicity of the peptide. You always get decreased retention times in reversed phase LC the higher the number of phosphorylations (with the exception sometimes of single phosphorylations and the use of certain LC columns)...

Hi Kostas,
I disagree with you and can prove my statement with a series of data that increasing the number of phospho groups in tryptic peptides with identical amino acid sequence will increase their hydrophobicity. If you use a default separation system for reversed phase LC/MS with a C18 column and mobile phases with acetonitrile and water + formic acid (0.05-0.1% FA), the result is late elution of multiple phosphorylated peptides and earlier elution of unphosphorylated ones and those with 1 and 2 phosphorylations. Tryptic peptides all do have at least one positive charge on amino groups under separation conditions mentioned above. The carboxyl groups will be protonated under that conditins, however you cannot protonate the phospho group. The charge of the phospho group "added" to the positive charge of the amino group will neutralize the overall charge of the peptide and it will be more hydrophobic. There are some peptides, but very rarely, where the negative charge of phospho groups prevail and they will be more hydrophilic in comparison to unphosphorylated ones.
Cheers

The zwitterion version is usually the most polar, for instance, of amino acids. Thus I would expect any compound with a negative phosphate and positive ammonium group to be more polar than a compound with only a positive or only a negative group. If a zwitter is more highly retained than its other versions I would think that invoking only the concept of hydrophobicity is a bit too simple.

That is true: zwitterion is more polar if alone and no other ions around it! However, in this case we have a kind of ion-pairing chromatography where the polarity will change depending on ion pairing agent used. There is a series of works form Mant and Hodgkes in Journal of Chromatography A.
Effect of anionic ion-pairing reagent concentration (1–60 mM) on reversed-phase liquid chromatography elution behaviour of peptides
Journal of Chromatography A, Volume 1080, Issue 1, 1 July 2005, Pages 58-67, M. Shibue, C.T. Mant, R.S. Hodges
Effect of anionic ion-pairing reagent hydrophobicity on selectivity of peptide separations by reversed-phase liquid chromatography
Journal of Chromatography A, Volume 1080, Issue 1, 1 July 2005, Pages 68-75,M. Shibue, C.T. Mant, R.S. Hodges