Chromatography Forum

Hello

I was wondering if anyone has had an opportunity to try Waters 'charged surface hybrid' columns. The basic idea being that they place some charge on the surface in order to improve the mass loadability and peak shape of charged analytes.

I was wondering if anyone has tried them. How well do they work? And do they work equally well with both anionic and cationic species.

Thanks in Advance

We have two in-house (C18 and phenyl-hexyl, I think), we have not yet tried them...probably won't for my next anticipated project, but perhaps for what may come two projects from now.

Hopefully someone else will have a better answer than I.

This was attempt of Waters to create analog of modern mixed-mode columns. Loadability and peak shape is better than regular C18. Mixed-mode still offfer better peak shape and higher loadability.

That's interesting.

I have heard (and read) a lot about mixed mode columns over the years. But I did not know that they were supposed to give better peak shapes, and better mass loadability, when doing reversed phase separations of charged molecules.

What is the reason for that exactly. Can you explain?

Thank You
Mark

With mixed-mode you have stronger cation-exchange group on the surface, with higher concentration of carboxylic groups. Both of these contribute to much higher capacity/loadability which is good for analytical and prep approaches. This groups shield analyte from interacting with your analyte.

Here are few references related to this issue:

A study of retention and overloading of basic compounds with mixed-mode reversed-phase/cation-exchange columns in high performance liquid chromatography Nicola H. Davies, Melvin R. Euerby and David V. McCalley 2007 Journal of Chromatography A, Volume 1138, Issues 1-2, 5 January 2007, Pages 65-72

http://www.sielc.com/Technology_Prepara ... raphy.html
http://www.sielc.com/Products_Obelisc.html

here are links for peak shapes:
http://www.sielc.com/Application-Improv ... olumn.html
http://www.sielc.com/Compound-Cetylpyridinium.html

It makes perfect sense that a negative charge on the stationary phase would increase loadability for bases.

What is interesting is that the Waters CSH columns actually have a positive charge. But still they have data showing that this is beneficial. I think McCalley also did a paper showing that either positive or negative charges on the column will help with loadability, and therefore the issue is most likely not a simple issue of electrostatic repulsion, but one of active sites on the column.

Vlad: Have you ever evaluated whether an anion exchange mixed mode column (so with positive charges on column) gives better loadability for bases vs a standard reversed phase column? I am curious what you found with such an experiment.

When you analyze basic molecules on anion-exchange mixed-mode you are operating in cation-exclusion mode (assuming that stationary phase and analyte carry the same charge) and capacity is increased by using MORE buffer. On RP you will still see tailing due to overloading and resolution will be affected by this. In case of mixed-mode (anion-exchange/cation-exclusion) basic group on the surface still provides shielding effect and you don't see effect of silanols. This is the main difference between SIELC columns and other columns. In Thermo/Dionex mixed-mode, ion-exchange group is far away from the surface and some of the analytes will "see" silanols. Imtakt is physically mixing silica gels, this approach effectively reducing capacity by at least factor of 2 (assuming that silica's mixed at 50/50 ratio).

Waters also says in one of there application note that CSH columns are better for peptide mapping and proteins to be used with less Ion pairing agents such as TFA

Quoting from this application note
"Using a nine-peptide mixture,it was demonstrated that the charged surface hybrid (CHS) C18 stationary phase offers greater peak capacity and, unlike most column chemistries,minimal dependence on strong ion pairing agents to obtain optimal peak capacity. this attribute suggests it is ideal for LC applications that require characterization using mass spectrometry."