Chromatography Forum

Alkyl, cyano, polymer, phenyl, etc, RP stationary phases offer selectivity differences that make natural ligand isolation using exclusively RP HPLC possible.

HILIC is nowhere near as celebrated as RP HPLC, however, there is also a diverse selection of HILIC stationary phases-- polyhydroxylethyl, cationic, zwitterionic, ERLIC, etc

Do these different HILIC supports provide significant selectivity differences that could conceivably permit multi-dimensional HPLC isolation of natural peptide ligands using only this mode?

As far as providing different selectivity (in the same mode), how does HILIC stack up against RP ?

Do these different HILIC supports provide significant selectivity differences that could conceivably permit multi-dimensional HPLC isolation of natural peptide ligands using only this mode?

Yes

As far as providing different selectivity (in the same mode), how does HILIC stack up against RP ?

Fewer columns are available for HILIC, so I would guess less variability for that reason alone. I suspect that the mechanism behind the selectivity variations are analogous in both cases. The primary retention mechanism in HILIC is arguably liquid-liquid partition between (relatively) water-poor mobile phase and a water-rich layer on the surface of the stationary phase. Once an analyte molecule gets into that water-rich layer, it can experience secondary interactions with the underlying column packing (adsorption, ion-exchange, etc.).

I would think you'ld see less selectivity differences from one HILIC column to another. Because, in HILIC, the primary stationary phase is an adsorbed layer of water. The "column stationary phase" can certainly have an effect, but I believe it is more of a secondary effect. Whereas in reversed phase, the column stationary phase is the whole deal.

Whereas in reversed phase, the column stationary phase is the whole deal.

Actually, I don't think it is. Aside from the whole issue of the "stationary phase" in RP actually being a solvated (organic-rich) surface layer, secondary interactions with residual silanols are a major contribution to selectivity variations among reversed-phase columns, which is part of the reason that you can seldom "plug and play" one C18 column for another. Hydrogen bonding effects (presumably at least partly with silanols) represent two out of the five parameters in Snyder's "hydrophobic subtraction" model of selectivity.

I think that there are significant differences. Let us start with silanols, which are readily accessible in HILIC and are beautiful ion-exchange groups without any drawbacks. You will have less of these things on bonded phases, so one of your retention mechanism is less effective with bonded phases. The ligands on the surface provide additional sorbtion sites for both a water layer and for the analytes themselves. Some of these bonded phases will be more effective with hydrogen bonding than others, for example. Then there is the classical game of retentivity, and you can get significant differences between the different phases that are available, which in turn will drive a selectivty influence of the mobile phase composition.
I can't compare this in a quantitative way to RP (lack of data), but my gut feel tells me that it is not bad at all.

In practice, I don't seem to be able to freely interchange a Luna amino column and a luna hilic column, so I assume they behave differently.

I've always worried about the idea that hilic is simply partitioning between a mobile phase and a sheath of bound water on a column. Problems with this model seem to me to be: (a) it doesn't explain why two hilic columns behave differently; (b) water is pretty small: how on earth can one end of the water be attached to a column and the other end just behaves like free water, so far as a much larger molecule is concerned? The larger molecule isn't free to gather a hydration sheath of attached water, in whatever positions it wants, because the water is also interacting with something (the hilic material).

Well, the fact that there are additional retention mechanisms available on HILIC phases does not mean that the partitioning mechanism into a water layer on the surface is wrong. There was publication by McCalley and me that demonstrated that the water layer exists on a silica column. Between 4% and 14% of the pore volume was occupied by bound water, so it is an appreciable amount.

Nice discussion. I believe the term "HILIC" implies hydrgen bonding to form a "water rich layer" allowing for partitioning to occur. But where in nature does hydrogen bonding occur? One example is in beta sheet on protein (meaning static interaction)

Chromatography is a dynamic process -so does H-Bonding really occur inside a column?

This is why we still prefer normal phase to "HILIC" and have replaced "Hydrogen Bonding" with "electrostatic interaction"

as for selectivity difference, just look at monosaccharides on NH2 vs any other normal phase or "HILIC" column

The term hydrogen bonding has been coined to indicate that there is something like a covalent bond formed, not an electrostatic interaction. The known number of examples in nature are enormous, for instance, that ice is lighter than water is explained via hydrogen bonding, the relatively high boiling point of acetic acid, etc. etc. etc.


Since I am in here now, I may mention in regard to the original question, that similarities between a silica column and silica based zwitterionic columns can be startling. Because of these similarities it is even more surprsing that there are also stark differences, for instance in the retention of chloride ion. My experience would indicate that basing selectivity guesses on the water layer are futile.

My experience would indicate that basing selectivity guesses on the water layer are futile.

No argument on *that* point!

But then again, *detailed* selectivity predictions on reversed-phase columns are essentially educated guesswork as well. The original question, however, was whether selectivity differences *exist* in HILIC, not how well we could predict them. Phenomenex has a nice example on page 15 of their Luna HILIC brochure (http://www.phenomenex.com/litlib/Luna/Luna%20HILIC.pdf). Same sample, same solvents, run on three different columns at two different pH values.

As in RP you definitely have multiple interactions. Besides partitioning, you have cation-exchange with silanols. For some of the bonded phases, for example for an amide phase, you have hydrogen bonding (yes, hydrogen bonding is not a solid bond, but something that can be formed and broken on the chromatographic time scale). For an amino phase, you have anion exchange with the amino function. The strength of the weak ion-exchange with silanols and amino functions changes with the organic content of the mobile phase which makes things even more fun. So there are a lot of things to play with. Read the paper by Grumbach, Diehl and me in the J. Sep. Sci.
HILIC is not dull at all...

The term hydrogen bonding has been coined to indicate that there is something like a covalent bond formed, not an electrostatic interaction. The known number of examples in nature are enormous, for instance, that ice is lighter than water is explained via hydrogen bonding, the relatively high boiling point of acetic acid, etc. etc. etc.

I'm pretty sure Hydrogon bonding theory is:

H-Bonding = (contribution from intermolecular electrostatic interaction only) + (contribution from sigma bond within the molecule)

The term hydrogen bonding has been coined to indicate that there is something like a covalent bond formed, not an electrostatic interaction. The known number of examples in nature are enormous, for instance, that ice is lighter than water is explained via hydrogen bonding, the relatively high boiling point of acetic acid, etc. etc. etc.

I'm pretty sure Hydrogon bonding theory is:

H-Bonding = (contribution from intermolecular electrostatic interaction only) + (contribution from sigma bond interacting with neighboring molecule)

Covalency of the Hydrogen Bond in Ice: A Direct X-Ray Measurement
Phys.Rev.Lett.82, 600-603:
http://prl.aps.org/abstract/PRL/v82/i3/p600_1

Summarized by the American Institute of Physics:
http://www.scienceblog.com/community/ol ... 00392.html

Maybe we should use 'Weak' H-bond to explain high boiling point of H20, ect.
and 'Strong' H-Bond to explain secondary structures of biopolymers, density of water at 4C, ect.

In terms of HILIC, all I wanted to say is The 'HILIC' model is difficult to conceptualize.
You have moving eluent (high kinetic energy),that is water soluble, that is flowing around
spherical particles. The packing material supposedly has such a higher affinity
for water that it retains a 'film of water.'