Chromatography Forum

I would even go further: the two charges in HILIC are VERY close to each other, such that they effectively cancel each other. Think about this in the following way: you would need to bring another ion closer to one of the bound charges than the three-carbon distance to get an effective ion exchange going on AND you have to have the counterion to the mobile ion around close enough to cancel the ionic repulsion for the bound countercharge.

When I played with the stuff (synthesized in our lab a long, long time ago), I did not see ionic interactions, but rather complete cancelling of all charge effects. This is why this zwitterionic stuff is also good for SEC. Of course, this assessment assumes that the material is prepared in a one-step derivatization, and that the silane is synthesized with the zwitterion in place before it is hooked up to the surface.

Uwe,
can you tell me or find one of your authors of the article on last Oct. LCGC issue about HILIC, that how to prepare ammonium formate in ACN?
when i mixed 0.1% formic acid with ammonia hydroxide in ACN they immediately precipitated out.
the article didnot mention how it is prepared, only to say during gradient run, " to maintain the constant concentration of the buffer"

I don't have the article here, so I can't answer your question specifically, but I can tell you how we usually would do this.

You prepare your mobile phase using 3 solvent channels. One solvent channel delivers acetonitrile, the other one water, and the third one the buffer. If you want to run from 90% acetonitrile to 50% acetonitrile at a constant buffer concentration, you run the buffer at a constant concentration of 10%, change the acetonitrile from 90% to 50% and make up the difference (0% to 40%) with the channel that delivers the water.

Going through the posts under this subject again, I thought, it might be worthwhile to clarify the mechanism of HILIC, at least the way I see it. I always thought of HILIC as a partitioning mechanism between the mobile phase and a surface layer enriched with water. Thus the stationary phase has no other function than to strongly bind water. The consequence of this is that it is difficult to get different selectivities from stationary phases that do nothing more but binding water.

The ionic interactions with amino phases or silica are secondary interactions. Neither silica or an amino group are strong ion-exchange groups. In addition, it appears that an amino bonded phase that is stable under HILIC conditions is a partially hydrolyzed phase, where the basic amino groups are for the most part neutralized by SiO- groups. The weak ion-exchange function of silica can be used with advantage to create retention for bases without any trouble, such as the excessive tailing common with older reversed-phase packings. Due to this, I prefer silica phases over the zwitterionic phases, but the zwitterionic phases work well for the purist.

a little argument here with Uwe,
in the presence of ammonium formate( i didnot measure the pH, but i will), wherever the amino get protonized if the pH is way below 7, i don't believe the silanol group will exist as SiO-, it should be in the most of SiOH; and if the pH is higher than or close to 7 where Sio- exists, the amnio group will be deprotonized and mostly neutral. so the ionic interaction between will be weak if present.
however, the interactions could still exist, but from the hydrogen bond part of N-H-O, and if this true, how to manipulate this for separation i am not clear.
do this make any sense?

What Uwe says, regarding ion interaction, also makes sense, but on the other hand, a zwitterion is usually highly polar....that just begs for interaction with ions. I just wonder what Ba++ would do in Patrick´s columns.
I am still dreaming of further improvements in silica based columns without any silanol interference, here we go again: optimized for one type of interaction (please don´t construe this again as a call for a general, do it all, column), so as to be able to separate compounds which are very close in structure, etc. Large selectivity differences seem to be good for SPE or speciality applications.

The pK of most silanols on a high-purity silica is around 7. On the older silicas, there are plenty of more acidic silanols around, which are for the most part missing on a high-purity silica. Consequence is that there are plenty of ionized silanols at pH 6, and some even left at pH 5 and less.

The pK of an aliphatic amine group is typically around 9 to 10. Therefore there is plenty of opportunity for ion-exchange on silica, even with the shift of pKs with organic solvent.

HW: I'll bet 2 beers that you will have a hard time finding silanols on a (well designed) zwitterionic phase with standard organic molecules.

the typical pH range HILIC runs is about 3-5, so i would think most silanol group will not be ionized. since it's already 2 units below its pK. the chance of SiO- is not that great, don't you think?
in fact the artilce by waters people on LCGC for silica HILIC just shows data run at pH 3-5. it looks really hard to convince me that at pH 3 there is strong SiO- presence.
here is the link to the article:

http://www.lcgcmag.com/lcgc/article/art ... ?id=126190

So???

The first time I ever did silica with aqueous-organic mobile phases I used a pH 7 phosphate buffer. Sample: doxepin, amitriptyline and related bases. Worked like a charm.

I agree that I do not expect silanol ion exchange at pH 3 for a high-purity silica. On the other hand, for an assessment of the influence of ion-exchange for a lower-purity silica, one would need to look at the data that were measured by Mendez, Roses and Bosch in a recent publication.

If you want ion-exchange, you will definitely get it at pH values larger than 5 even for a high-purity silica and possibly to weaker level at pH values below 5. Which is what I said before...

Smiley,

A quick answer to you question about where to apply HILIC.
I’m still getting puzzled when I realize the vast applicability as it may be used in many cases, but to generalize we tend to think that a chromatographer should apply HILIC for those compounds that do not, or have little retention on a reversed phase column using standard conditions. Compounds that have poor selectivity in reversed phase systems but differ in their hydrophilic functionality are also suitable candidates, if they can be ionized. A good example is flavonoids…

Smiley,

We and collaborators at the academy is working on fundamental comparison among different HILIC phases, and it will be presented at HPLC 2005 in Stockholm.

Regarding the availability of our polymeric columns in US, please contact our distributor Alltech again. They do not carry all our products in their catalogue, but you should be able to order any dimension through them anyway. Another alternative would be to place and order directly to us in Sweden. All necessary information may be found at www.sequant.com

Uwe, ok, since you didn´t say to forget all but standard organics...

Patrick, et. al., Am still wondering about ion exclusion in zwitter columns.

Just had a look at the bible of TLC, Egon Stahl´s Dünnschicht-Chriomatographie. Should be renamed TLC and HILIC?

HWM- I agree. There are a lot of forensic TLC separations of drugs of abuse done with a mobile phase containing 100% methanol and a dash of aqueous ammonia-this must be a HILIC type separation.

In support of Patrick's comments regarding the unique character of the retention mode for zwitterionic materials, there is quite a bit of literature demonstrating the fact that zwitterionic media interact with electrolytes in a manner which is distinct from ion exchange processes. You can find most of this information in conjunction with study of adsorbed zwitterionic surfactants on both silica reversed phase and polymeric reversed phase media. A theoretical discussion of the retention mechanism can be found in at least two papers authored by Paul Haddad in the Journal of Chromatography.

Regardless of the theoretical underpinnings of the retention mechanism, several experimental observations clearly demonstrate the unique character of this retention mechanism. First and foremost is the inverse relationship between retention and ionic strength for ionic analytes relative to the conventional relationship in ion exchange. That is: retention of ionic analytes actually increases with increasing ionic strength (at least up to a point) rather than decreasing as is the case for ion exchange retention. This clearly demonstrates that the retention mechanism is not ion exchange but better described as "salt exchange". Secondly, using this technique it is possible to obtain for separate resolved peaks when injecting a simple mixture of two salts (i.e. all permutations of anion-cation combinations have unique retention times). Some proponents had proposed this might allow useful separations of ions with water as the mobile phase but exceedingly poor chromatographic performance is obtained under these conditions. To get good chromatography in conjunction with such materials (at least for a small ions) it's important to have at least some all electrolyte in the mobile phase.

As you can see from the above, these chromatographic properties are quite unique when compared to other stationary phases. For that reason, I question the wisdom of lumping this stationary phase type into the generic category of HILIC since control parameters are clearly different from other HILIC phases.