Regeneration techniques for HILIC silica columns

[Kazimierz]

I'm interesting what regeneration method chromatographers using for siilica columns in HILIC chromatography.

The main advantages in HILIC is that many class drugs could be use on one type of column and mobile phase (in reverse phase only one type of drugs).

Also, in HILIC, efficiency is >40000 plates/column=>160000 plates/meter. Additionally, event without extraction, impurities from blood plasma do not represent special problem after acetonitril precipitation only. Therefore, with such method correction of drug-dose is possible even in 30 minutes.

Actually, I'm using recirculation with 100% acetonitril for column regeneration overnight, every week. When column do not works, mobile phase is flowing 0.2 ml/min all time. Column works since 4 month on this same precolumn without any lost efficiency or retention-time.

[MG]

I too would like to hear what people are using. I have used HILIC in only a few special cases for qualitative analysis, so I have not yet had problems with the columns wearing out. I am surprised that 100% ACN works well for regeneration. I would think that a high water content would be needed to remove strongly retained contaminants.

On a slightly different subject, a reason I have not been anxious to develop HILIC methods for quantitation by LC/MS, is that I don't have a good understanding of how salts (e.g. from biological fluids) behave in HILIC. Are they strongly retained only to bleed off during gradients after many samples, or are they weakly retained or unretained as in reverse phase? In reverse phase, one can inject samples with a high salt content and simply divert the void volume to waste, and signal suppression or ion source fouling due to salts are easily avoided.

[maris]

For bare Silica HILIC columns we use diluted sulfuric acid solution , then water to neutral reaction followed by organic solvent.

[Uwe Neue]

A good way to wash silica columns used for HILIC is a strongly acidified solution of roughly 50/50 water/organic. The acid breaks the interaction with silanols, the high water content eliminates HILIC, and the organic content prevents hydrophobic interaction with siloxanes.

To MG: salts do not present a retention problem in HILIC, but VERY high salt concentrations would cause precipitation problems due to the high organic content of the mobile phase. If you do the sample preparation of plasma samples with SPE, there is no salt left to speak of. If you are running a HILIC gradient on a plasma sample after protein precipitation with acetonitrile, I do not see a problem either.

[MG]

Uwe, let's take a simple example of salts in HILIC. Suppose my mobile phase is 90:10 ACN/H2O w/ ammonium formate, isocratic. Column is silica. Suppose I have a sample, dissolved in 90:10 ACN/H2O, that contains a significant amount of sodium chloride. Will the NaCl elute in the void (as with reverse phase)? Or will it have some finite retention due to some sort of HILIC interaction? Or will only the Na+ be retained due to ion exchange? Yes, I could do the experiment myself, but I am hoping you already know the answer!

[Uwe Neue]

Actually, I do not know where it would elute. You don't see it in the UV, our MS people have not complained either. I suppose somebody's gotta do some exepriments.

[HW Mueller]

I am playing with some radioisotope ions, mostly reverse phase HPLC, but also some normal type of TLC. Some surprising results can be had. In your example, MG, I would venture a guess that the Na+ (and Cl-) would be flushed out by even very low conc ammonium formate at pH where SiOH dominate. For pH where SiO- dominates you may need a higher concentration of ammonium formate to get NaCl out. You may get problems if sample is dissolved in anything other than mobile phase. If you had only 10% H2O in the mobile phase? .... I pass also.

[RA Koster]

I'm setting up an lcms/ms method fot the determination of a zwitterion with a relative low mass (200 amu).
I'm now trying a Hilic silica column, but because it is an zwitterion i can't get the best retention.

About the questions what happens with salts, i can answer that for my situation. I have tested suppression today and it sucks:D
It has practically no retention but it's quite wide and my compound has got about the same retention as as the suppression.

The sample preparation is SPE.
Mobile phase is acetic acid 0,1% : MeOH 70:30
I have tried ACN and that has better retention as expected, but the sensitivity is much lower.

Does anybody have experience getting retention with an zwitterion on a ZIC pHilic for example? Or with adding HFBA as an ion pair to a zwitterion?

[Uwe Neue]

The addition of an ion-pair reagent is (usually) counterproductive in HILIC

[MG]

It is not surprising that you get little retention with 30% MeOH and the balance aqueous. Normally HILIC is done with 50% or more organic, and methanol will be a stronger eluting solvent than acetonitrile (but weaker than water).

[crlar]

RA Koster,
At the present, I am working in two molecules simmilar to yours (zwitterion, low mass) and for the moment, my best results in chromatography have been using HILIC. But in both cases, the mobile phase is 90-95%ACN:10-5% buffer at pH 5.4. I work in HPLC-MS/MS (ESI negative) and I would have more sensitivity at higher pH, but 6 is the maximum for the column.
On the other hand, I have tested that increasing the % of water and lowering pH (as you propose) I have a high loss of sensitivity. Don't you?

We are studying the possibility of testing p-HILIC in order to increase the pH, but we haven't done it for the moment

[Einar Ponten]

The need for higher pH in some MS applications has encouraged us to develop the polymeric ZIC®-pHILIC column. The zwitterionic phase has a zero net charge regardless of pH and mild ion exchange properties. Moreover, the polymeric material can withstand high pH.

Using native silica a raised pH will increase the ion exchange properties and discriminate the HILIC mechanism. It means that a very high ion strength (buffer concentration) is required for elution of some analytes. An unfavourable situation in MS detection!

[RA Koster]

crlar

When i use a high percentage of aqeous mobile phase i have a extreme loss of sensitivity too, as expected.
I am going to try negative ionisation, then a ZIC-pHilic column seems to be suited.
Are you using Turbo ESI? I'm using TESI with a API 3000.
What kind of retention times do you achieve?
And have you tested suppression yet?
I'm very interested in your test results, using the ZIC-pHilic column.

The component i'm analysing is very similar to Dopa (one amine+ more acidic groups).

Correction:

Mobile phase is acetic acid 0,1% : MeOH 30:70 (sorry)

[crlar]

Yes, I am using turbo ESI with an API 365.
Our happy results with one of the molecules using HILIC (silica) wheren't so good after we tried to reproduce them after some days: the retention times shifted, the molecule is not retained and the peak has an important tailing...we traid to "regenerate" the column (thinking we have had a contamination problem) but we are not getting the same retention times and shapes any more...
At the present we are checking if we are able to reproduce the results of the other product...

On the other hand, I have been inquiring about the "explanation" for the loss of sensitivity with increasing the aqueous content of the mobile phase in negative ESI but for the moment I have no answer, and I have some publication where some of my molecules are analysed in negative mode C8 or CN columns with high acqueous content...do you know if the API 365 and API 3000 turbo ion sprays have a really different sensitivity to this phenomenon than other instruments? Why?