Diol Columns for Separation of Proteins/Peptides

[adam]

Hello All

I was just looking through the Thermo Scientific Chromatography catalogue.

They have a Diol column which they state is "for reversed phase separations of proteins and peptides".

I've done a fair amount of protein/peptide chromatography over the years but I have never heard of using a Diol phase for reversed phase separation of these compounds. Can anyone offer anything on this. Is there a certain niche where this would be used or...? Is it used when C4 and C18 don't work...?

I would like to understand this better. I would also be interested in any publications that discuss this.

Thanks very much

[DJ]

This has to be a hilic column.

[Jack Silver]

Although HILIC is a possibility, I've seen some applications where they use a diol column as a size exclusion column.

YMC markets such a column here:
http://ymc.co.jp/en/columns/ymc_pack_diol/

[DJ]

Andy Alpert has a PHE Hilic column with SEC application notes. In principle, couldn't -any- column of like pore size be used as SEC? I'm not sure why HILIC columns are used. Perhaps it is because peptdide*stationary phase binding can be thoroughly prevented with appropriate mobile phase.

[adam]

Thanks for the replies guys, but actually they are using a diol column as a reversed phase separation.

Anyone heard of this?

Thanks

[Andy Alpert]

While I haven't heard of an example of this before, and it's an unlikely combination of column and mode, it's not impossible. With modes of chromatography that involve a partition mechanism, you can get retention of an analyte provided that the difference in relative polarity of stationary and mobile phases is great enough. For example, with a regular C-18 material, water suffices. With the mildly hydrophobic materials used for HIC (hydrophobic interaction chromatography), you have to add something like 1.6 M salt to the mobile phase to attain that difference in polarity. My paper that introduced HILIC (J. Chromatogr. 499 (1990) 177) goes into some detail with this regarding uncoated silica, which has been used in both the HILIC and reversed-phase modes.

Do I think it's a good idea to use a diol material for reversed-phase chromatography? No.

[danko]

The term reversed phase is derived from the hydrophobic properties of – yes - hydrophobic materials like C18, C8 etc. Now, diol is actually a very hydrophilic entity and is typically used for eliminating the weak hydrophobic property that silica exhibits especially when utilized in silica based SEC stationary phases, in which mode the eluent is often 100% aqueous and thus no organic modifier present to eliminate the hydrophobic interactions.
So, if diol is mentioned as a reversed phase entity, I would expect it to be a simple error.

Best Regards

[mauro]

hi Adam,
this must be a mistake. diol columns are for SEC of proteins. I'm looking at at Thermo catalogue 2012-2013 right now, but i don't find the same statement you found. can you point out where it is?

thanks
Mauro

[Jack Silver]

In my experience, diol columns can be run in normal phase (either with the common normal phase solvents or as HILIC), or as a reverse phase column. Non-polar run in aqueous solutions on diol columns tend to run as reverse phase. Polar compounds tend to run as normal phase from 100% organic to roughly 50% aqueous (depends on the organic solvent).

Please see this link for examples:
http://www.isco.com/WebProductFiles/App ... havior.pdf

For information about using a diol column in organic solvents, please see:
http://www.isco.com/WebProductFiles/App ... _Phase.pdf

Disclosure: I wrote this app note & poster