IEX or HILIC for peptides - any experience?

[Kostas Petritis]

How about we compromise and say that HILIC is orthogonal for peptide separations (according to Gilar et al work) and complementary for small molecule analysis?

[Einar Ponten]

Well, I agree. There is nothing as good as real data, better than semantics...

Evaluation and Optimization of ZIC-HILIC-RP as an Alternative MudPIT Strategy
P. J. Boersema, N. Divecha, A. J. R. Heck, S. Mohammed
J. Proteome Res., 6 (2007) 937-946

By using RP and the orthogonal HILIC they got considerable better results than with other combinations, as also presented at ASMS 2007

[Mattias]

I will have to order these articles, I agree that real data is the way to change my mind.

I find it very hard to believe that a group of hydrophilic and a group of hydrophobic peptides will not form the same groups in both RP and HILIC (but in reverse order). If they do, the methods are by definition not orthogonal.

But once again, I have not read the articles so I have to keep my mind open.

[HW Mueller]

So, if one gets the opposite results it is 180° or double orthogonal. If one is satisfied with an orthoganal method one should have double the satisfaction here. Or is this not mathematics?

[Mattias]

...

Why not identical results and 360°C between the axes...

Back to the topic - I realise that it is probably impossible to find a method that is 100% othrogonal to RP. Even SEC and IEX columns always have some hydrophobic interactions going on. I might be so that HILIC is the best option for peptides - I honestly don't know. When it comes to results in publications I always think of a poster I have in my office (of how to read scientific papers): "Typical results are shown" means "best results are shown".

I'll have to test it myself in the lab!

[Kostas Petritis]

When it concerns the peptides, there is a lot be done and I have also worked on the problem a lot. From my perspective, strong cation exchange is the best if you would like to take a limited amount of fractions (i.e. <20 or so) from the first dimension.