Effect of Methanol in a HILIC Analysis

[Jimi]

Hi

So I want to ask if anyone has experimentally evaluated the effect of adding methanol to a HILIC analysis. For example, let's say we have an analysis with 95/5 ACN/Water. And then we try it with 95/2.5/2.5 ACN/Water/Methanol (in other words half of the water has been replaced with methanol. Some seem to suggest that this will result in increased retention simply because methanol is weaker than water (as an eluent). But others seem to suggest that the added methanol may become part of the sorbed water layer, and this would result in decreased retention.

My guess is that a combination of both would happen. But then it's not clear what the net effect would be. Has anyone tried it. There is a reason I am asking, but I am intentionally not going into all that for the moment.

On a related note, is it possible to do HILIC with no water at all, but with methanol as a replacement. In other words, if the mobile phase was something like 95/5 ACN/MeOH could we get a sorbed layer of methanol. Maybe addition of buffer would help here as it would further promote stratification of the methanol and ACN phases.

This one is for the true experts. What do you think folks.

Thanks very much.

[Jiri Urban]

Colleague of mine tested it, you might find more information in J. Sep. Sci. 2013, 36, 2753–2759 (if you do not have access let me know and I'll send you the paper).

[HPLCaddict]

From the column use and care manual of a certain Waters HILIC column:
"Alternate polar solvents such as methanol, ethanol or isopropanol can also be used instead of water to increase retention".

As a personal addendum, I once used a mobile phase of acetonitrile/methanol 90:10 successfully on a HILIC column with a compound that showed great hydrolytic instability in water mixture...

[carlo.annaratone]

The article on J Sep Sci seems very interesting... I will be reading it accurately. Thanks for highlighting it

[M Farooq]

So I want to ask if anyone has experimentally evaluated the effect of adding methanol to a HILIC analysis.
On a related note, is it possible to do HILIC with no water at all, but with methanol as a replacement. In other words, if the mobile phase was something like 95/5 ACN/MeOH could we get a sorbed layer of methanol. Maybe addition of buffer would help here as it would further promote stratification of the methanol and ACN phases.
This one is for the true experts. What do you think folks.
Thanks very much.

There are couple of interesting points here. We routinely do use pure methanol and ACN in "polar organic mode" on highly polar hydrophilic columns. We do not call it HILIC. Generally, the retention is increased when methanol is used and peak shape is broadened. It may be a great approach to play with selectivity. One can, in practice, make as complicated mobile phase as we want why not stick to a simple mobile phase? There must be a reason to make a complex eluent.

Chromatographers should keep in mind that HILIC is essentially a mixed mode separation; if it were simply a partitioning of the analyte in the (proposed but perhaps not proven) stagnant water layer, there would be no selectivity difference on any HILIC column. All the analytes would elute in the order of their solubility in water (or better octanol-water partition coefficients) on each and every HILIC column. This is not the case.

Dr. Alpert (the man behind the HILIC word) is here and he can give you a better idea, if he reads this post.

[Klaus I.]

On a related note, is it possible to do HILIC with no water at all, but with methanol as a replacement.

Yes it is, but I think this is called 'normal phase'?

[Jimi]

Thanks Klaus, but actually this is not what I mean.

What I am asking is whether you can do HILIC with methanol instead of water, such that the methanol forms a sorbed layer on the column just as would happen with water. So this would not be classical normal phase, it would be in the HILIC family.

Thank You
Jimi

[tom jupille]

So this would not be classical normal phase, it would be in the HILIC family.

I think this may be a "distinction without a difference". The broad definition of "normal phase" is a separation wherein the stationary phase is more polar than the mobile phase. By that definition, HILIC is a type of normal-phase partition chromatography.

such that the methanol forms a sorbed layer on the column just as would happen with water.

It probably does, at least to some extent. What you have to remember though, is that the "sorbed layer of water" is a model (a mental picture, if you will) of how HILIC works. I like to quote from the statistician George Box who (admittedly in a different context) said "All models are wrong. Some are useful." If I had to speculate, I'd guess that methanol *does* preferentially sorb to a polar column packing to some extent. I'd also guess that any "sorbed layer" is less distinct than it would be with water. And I would certainly argue that any distinction between "HILIC" and "polar organic mode" is more a matter of semantics than of substance (e.g., what happens if you have water *and* methanol *and* acetonitrile?).