Chromatography Forum

Hello all,

I'm back to using HPLC again after a significant sabbatical away...

We're a low throughput lab (with regard to HPLC quants at least) and we get a lot of one-off requests. I'd like to explore the potentiality of HILIC as it will be another technique we could use for the polar/ionic compounds.

Reading through the literature it seems that bare Silica HPLC columns can be used for HILIC with aqueous RP mobile phases e.g:

http://www.sigmaaldrich.com/etc/mediali ... 10093h.pdf

http://www.labplus.co.kr/tech/uploadHTML/HILIC.pdf

Is this true? I'd always presumed that a specialist bonded phase was necessary.

In the dim dark past I've never run anything more polar than neat IPA through a bare Silica column. I also remember from my graduate student days that when I ran a flash column with more than 40% MeOH/DCM I'd get a white precipitate (which I presumed to be Si) which I'd have to filter from my purified natural product. I realise a bonded phase is different from the TLC grade Si we'd use for our homemade flash columns but all the same...

I have a donated Hypersil Silica 5um (4.6 x 250 mm) column that can be sacrificed if the need be. Can I give this a go, or will the stationary phase be stripped off?

Just wanted to ask before I have a play around and attempt to get a feel for this technique.

Many thanks in advance.

The basics of HILIC:

- it essentially depends on formation of a water-rich boundary layer due to hydration of a polar stationary phase. If this is effectively more polar (has a higher water content) than the mobile phase, then analytes can partition between the two phases.
- it seems to work best with acetonitrile as the organic part of the mobile phase
- in principle, any stationary phase that is sufficiently polar to hydrate well can be used. Such phases include silica, diol, amino, polyhydroxyethylamide (the "original" HILIC column), and various proprietary "zwitterionic" phases. Each of these can give different selectivity, depending on secondary interactions.

You're on the right track trolling the manufacturers' literature for suggestions and applications; there's a lot of information out there.

A good general starting point is a gradient from 5% to 45% water (or buffer) in acetonitrile (remember, this is effectively normal phase chromatography, so water is the strong solvent!).

edit: I originally had the percentages going the wrong way!

Quick start eluent for HILIC is 70% acetonitrile 30% 50mM ammonium formate or acetate, with a silica column you almost have to go low in pH so 3 to 4. For very hydrophiic compounds or compounds with multiple ionic interactions you may have very long retention with this eluent.

Thanks for the replies people.

I'll give your suggestions a go and see how we do. Its still playing with my head a bit using reverse phase mobile phases on normal phase columns. I'm sure I'll manage to adapt...

passy,
There are few fancy names for this chromatography like hillic and reverse phase reverse chromatography.
Just treat it as normal phase chromatography, eg original Tsvet’s experiment of separation leaf pigments on chalk powder. Is not that great to have ref. in your work to paper dated 1890th!
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"If your experiment needs statistics, you ought to have done a better experiment." Rutherford