Advantages and disadvantages for HILIC columns ....

[JOCAPEN]

I am sorry friends...but I dont have experience for this column, but I have plans for this column....please, Could you write here yours expectation and problems....

Tanks a lot

[Bryan Evans]

What type of compounds will you be working with?

[goxy43]

For me, separating peptides and some small basic substances like serotonin and 5-HIAA the column works perfectly with different mobile phases used. But, as already mentioned, you should specify what type of separation you intend to perform. Since the starting condition for HILIC is around 80% AcN you should be careful if there is any danger of precipitation for your sample.

[JOCAPEN]

My target is a polar compounds..amino acids, carboxyl acids and other....now I have been used monolithic column...some times ion pair for RP (spherical)....

tanks for everybody

[Einar Ponten]

The fundamental principle is to separate orthogonal to reversed phase (RP). We always have to underline that HILIC is not RP, otherwise it is hard to enjoy the benefits and to do the proper things.

Basically, any compound that isn't retained on a RP column will be on a HILIC column. However, these are very different and therefore it is adviceable to consult the support of the supplier for any HILIC column before starting.

We have developed a tool that gives an initial idea on the suitability of a ZIC®-HILIC column, please check the "HILIC Prediction" at http://www.sequant.com/prediction

The fact is that many compounds can be separated both in HILIC or RP mode, and the significant benefit with HILIC is that you typically increase the sensitivity while using MS or ELSD.

[Uwe Neue]

Many polar compounds can be retained on a reversed-phase packing like Atlantis T3 that gives strong retention in a purely aqueous environment. Most RP columns can't do that.

HILIC columns like Atlantis silica often give very good retention (together with other benefits of the technique), but you need to relearn, how to execute this type of chromatography and what to avoid.

HILIC commonly gives you better MS sensitivity than "normal" reversed-phase.

In HILIC, water is the strong solvent. Analytes that retain best in HILIC also are easily dissolved in water. Injecting your HILIC sample dissolved in water is the same as injectiong a reversed-phase sample dissolved in THF or DMSO. Injecting your HILIC sample in water is asking for trouble.

[emsnyder]

Not much to add other than I like to use HILIC.

I do have a pet peeve, though. HILIC is not orthogonal to RP. "Orthogonal" is overused. Mathematically, it means perpendicular to. When used in the context of chromatography, it means that the separation is based on different characteristics of the molecule. RP and HILIC are both based on hydrophobicity, just opposite each other. (I know, there are secondary mechanisms that affect selectivity, but the primary characteristic is hydrophobicity for both). People will also use the word orthogonal to describe different RP bonding chemistries.

An orthogonal method to RP is ion exchange. It's primary mechanism is charge state.

Another would be size-exclusion - based on size.

I really find HILIC useful, but it is not "orthogonal" to RP.

[lmh]

emsnyder, Absolutely! Yes! I'm glad someone said it.

Just to underline Uwe Neue's point about injecting in water, do remember too, that if you have a loop injection system making injections where the loop is only partially filled, you need to look at the needle wash solvent, or whatever other solvent is used to fill the remainder of the loop. There's no point in injecting 5uL of carefully considered methanol-dissolved sample if it is surrounded by 15uL of very aqueous needle-wash solution used by the injection system to make up volumes in the loop.

[HW Mueller]

To me orthogonal in this context means complimentary. IX can also be complementary to RP, etc., but that IX is 90° to RP while HILIC is not, is difficult to see. All are due to electronic interaction. Certainly I would consider it as convincing if one got the same results comparing RP to HILIC as if the person compared RP to IX, etc.

[Uwe Neue]

There is a paper in Anal. Chem. 77 (2005) 6426-6434 by Gilar that addresses the question of orthogonality of methods in a simple and quantitative way. With respect to the current discussion, he found that HILIC on silica was orthogonal to RP, at least for his peptide samples

He also looked at SEC, IEX, high-pH versus low-pH RP, etc. A very good paper!

[emsnyder]

I can see how that would be in that case. I would suppose that there is a lot of IEX behavior of a silica HILIC column with peptides.

I would think this would be less for bonded-phase HILIC columns, as well as for compounds that are "less" cationic.

Most of the HILIC applications I have seen have the reverse elution order from a RP column. I would assert that any "pure" HILIC separations are opposite RP. Where secondary interactions are significant for either column, they would tend to be more orthogonal.

[Kostas Petritis]

In order for SCX to be completely orthogonal to RP for peptide separations you need to eliminate secondary hydrophobic interactions by using about 25% acetonitrile. By doing so, I got a correlation coefficient R2 of 0.001 when plotted 65,000 peptides SCX elution time to RP elution time... (an Anal Chem paper will be out on this in the coming months...).

[lmh]

Wow! HW Mueller, I think I like your idea of complimentary best.

Do I get the impression part of the debate here is because two different groups of people are using Hilic for radically different purposes? The proteomic people want it to be a 2nd dimension, so they correctly worry about orthogonality. The small molecule people are turning to Hilic as a fall-back when RP fails, so they don't care at all whether it's orthogonal. In fact they like the idea of it being a similar axis to RP, but the other way round.

Also, orthogonality is only visible if you have a wide enough range of points. The small molecule people are working with a far more diverse cloud of analytes than the protein hydrolysate people, so they might see inverse correlations that don't show up in the narrower range of possible peptides. This doesn't matter of course: the peptide people only need to know that hilic appears orthogonal to RP over in the region of "chemical-space" they are using.

[Einar Ponten]

I guess I started the sematic discussion, but anyway everybody seems to agree on the potential of HILIC.

In response to the last post. There is a trend to start to test with HILIC and if that fails, then use RP.
The rationale is that by using HILIC it opens for smoother and faster sample preparation and increased ESI-MS sensitivity (mobile phase composition and potential to avoid suppression effects).

Relating to the heading, any HILIC disadvantages?

[XL]

There is a trend to start to test with HILIC and if that fails, then use RP.
The rationale is that by using HILIC it opens for smoother and faster sample preparation and increased ESI-MS sensitivity (mobile phase composition and potential to avoid suppression effects).

In fact, it is genrally true that try RP first, then HILIC if RP fails. Above statement could be true ONLY if you are dealing with highly hydrophilic molecules. If so, the selectivity of a HILIC phase might not be right. By comparison, RP is more versatile for both hydrophobic and hydrophilc molecules.

Also I would like to point out that many stationary phases can be used as HILIC columns, such as bare silica, amino, diol, amide, polyol, zwitterionic and reversed-phase and ion-exchange mixed-mode phases. The different polarity results in different selectivity.