HILIC column

[jusuom]

I have two questions, which somebody might be able to answer:

1) How does the stationary phase of a silica HILIC column differ from the ordinary Si column meant for NP-LC?

2) How to recover a Si HILIC column if one for some reason accidentially washes the adsorbed water layer away with e.g. 100% organig solvent?

[Andy Alpert]

In response:

1) It's the same column. Starting in 2003, some companies saw fit to relabel their normal-phase silica columns as HILIC columns, since the market for HILIC applications was growing. Switching from one mode to the other merely requires a change in mobile phase.

2) Just elute the column with a solution that contains at least 10% water and a polar column material (including uncoated silica) will adsorb water to form that immobilized hydration layer. This is analogous to the water in a molecule of K2HPO4.3H20; it's an integral part of the molecule that associates with it more strongly than any water molecules in the rest of the solution. In general, one should have at least 2% water in the mobile phase in order to replenish the immobilized aqueous layer in HILIC.

I might mention that materials with a thick, well-hydrated coating can outperform uncoated silica for many applications in HILIC. These are harder to make than uncoated silica, which a packing shop can pack into a column with no synthesis at all. That accounts for the enthusiastic advertising of uncoated silica columns by some companies.

[jusuom]

Thank you for your answer. So does this mean that I can use any normal Si column (like Restek Pinnacle DB Si column) as a HILIC column after appropriate treatement with aqueous solvent?

[Andy Alpert]

Yes. Just be sure to elute the column for at least 40 minutes with the starting mobile phase for HILIC if you want to get reproducible retention times for the most weakly-retained analytes. It takes longer to reequilibrate a column in the HILIC mode than in most other modes.

Just because a column can be used for this mode doesn't mean that it's the optimal one for the purpose...

[jusuom]

Thank you very much, Andy. Obviously you have some concerns on the usability of HILIC Si columns?

[Andy Alpert]

They're fine for something undemanding like analysis of a drug or other small molecule, which describes a large fraction of HPLC analyses. They are not fine for HILIC of peptide digests, intact membrane proteins, etc. They are also inferior to materials with thick, well-hydrated coatings in cases where an analyte is poorly-retained in HILIC or is only marginally soluble in the mobile phases needed for retention (e.g., an oligosaccharide in 80% ACN). The better hydrated a stationary phase material is, the less organic solvent is necessary to get a given degree of retention of an analyte in the HILIC mode.

Silica columns are cheap and simple, and can be more durable under some conditions since they have no coating to lose. If they work adequately for a particular application, then by all means go ahead and use them.