Retention (or elution) of sulfonic acid

[Mattias]

I have an impurity that I want to run on LC/MS. The impurity is extremely hydrophilic and negatively charged in all organic acids (even in 100% formic acid). The structure is unknown, but I have reason to believe that it must contain a sulfonic acid. In the LC/UV method we use triethylamine and the impurity has good retention. But I am not allowed to use triethylamine in our QTOF...

1. I have tested Waters Atlantis T3 and 100% aqeuous - no retention of the impurity.

2. I have tested anion exchange (Primesep D), but I have not been able to elute the compound with reasonable amounts of NH4OAc (up to 100 mM).

3. I have tested HILIC (or at least similar) with a Zorbax NH2 column and gradient from 90 - 50 % ACN. I have a feeling that my impurity must have precipitated on the column since the pressure went sky high after injection.

(BTW, my impurity is dissolved in 50% MeOH, but I only inject a couple of µl so that should not be any problem)

I am glad for any suggestions for how to get retention of this compound with LC/MS friendly conditions.

[Uwe Neue]

If it is as polar as you think, HILIC must be best. But do not inject an analyte with a sufonic acid group on a NH2 column - ypou will never see it again. Plus, I would not put a NH2 column in front of an MS, unless I plan to spend a few weeks cleaning it.
Use a (Atlantis) silica HILIC column for this analysis instead.

[Bryan Evans]

If your solute precipitated at high organic, why waste your hard earned cash on a silica column?
You can try Scherzo SM-C18 (C18 + anion ligand + cation ligand). Below are organic acids done using RP + IEX:

http://www.imtaktusa.com/site_media/fil ... TI518E.pdf
http://www.imtaktusa.com/site_media/fil ... TI542E.pdf

NH2 phase may require TEAA buffer for this solute. Unison UK-Amino can be used with ELSD, CAD, or MS.
No problem whatsoever with bleeding.

[Vlad Orlovsky]

You need a short Primesep B2 column, or you can try Obelisc N in ion-exchange mode similar to these. Obelisc N can work at differnet pH (2-5) and give you differnet retention. At different pH yo change ionization of acididc fragment of Obelisc N column, which can REDUCE anion-exchange happening on basic site of the stationary phase.
http://www.sielc.com/compound_315.html
http://www.sielc.com/compound_405.html
http://www.sielc.com/compound_393.html

[Mattias]

Thank you all for your suggestions!

I am reluctant to move to HILIC after the "high pressure alert" I got from injecting the compound in a 90% ACN flow (it went from 70 to 400 bars in seconds).

Some kind of "switchable" anion exchange column would be best where I just increase the pH to elute the compund. I will look into the options presented.

[Vlad Orlovsky]

we run naphthalene disulfonic acid on Obelisc N (4.6x50 mm) column and retention drops from 12 min at pH 3 to 2 minutes at pH 5. This happens due to change in ionization state of stationary phase and is only possible on true mixed-mode column were both ionic groups are on the same molecule. Obelisc N is zwitter-ionic phase with positive and negative charges separated by a linker.This approach will not work on the mixture of silicas (Scherzo SM-C18) because basic and acidic groups are on different silica particles and the phase it not zwitter-ionic. In zwitter-ionic phases you can change relative strength and equilibrium between basic and acidic groups of the stationary phase if the linker allows this.

I found two sulfonic dyes and we will run it next.

[Bryan Evans]

Hi Mattias -

Sorry but we do not have any data with sulfonic acid. As you said, the
challenge will be getting this thing off the cationic ligand.

Vlad -

Sorry but we have plenty of data with pH gradients.
We have data where RT of solute changes at different eluent pHs.
Works great!

[Vlad Orlovsky]

I have no doubts that pH gradients work with Scherzo SM-C18, after all phase has ionizable groups, but I don't see how this will work for disulphated molecule. You cannot significantly decrease ionization state of disulfate molecule (only increase the strength by going up in pH) and you cannot change IE properties of the phase, while decreasing IE properties of the analyte (very low pKa values). You can probably elute this compound from Scherzo SM-C18 but it will require at least 150 mmol of ammonium formate buffer at pH 2.8 (assuming that only 50% of sites of the blended stationary phase are basic) and peak shape is going to be ugly due to strong double inetraction. The task was to reduce retention by changing pH

[Bryan Evans]

I would not recommend ammonium formate at pH 2.8.
I would recommend 50-100mM ammonium formate (neutral pH).

If you would like to learn more about how the properties of multi-mode ODS, I would be happy to forward a poster on to you.

[Vlad Orlovsky]

At neutral pH both sulfonic acid groups are going to be fully ionized and unless Scherzo SM-C18 has basic group with pKa of 5 or less (modified pyridine), there is absolutely no way to elute disulfate from this column at reasonable for LC/MS buffer concentrations (less than 50 mmol). Also, I don't think that buffering capacity of ammonium formate is good at neutral pH (6-7)

[Uwe Neue]

To go back to Mattias's last concern:

In HILIC, it is always best to inject the sample from a solvent composition with a high acetonitrile content. You will see if there is precipitation, either of your analyte or from the matrix. If you see something, then this can be the problem for a pressure. Otherwise, it could be adsorption of your sample to the amino column, and this is not very likely to translate onto a silica column.

Most standard amino columns are problematic in HILIC. So I am not surprised that you saw problems with your column.

[Kostas Petritis]

You can also try a porous graphitic carbon column... it has been shown to retain even inorganic ions...

[Mattias]

Kostas> I have tried this also, but I didn't manage to elute the peak (or the peak was so terrible in shape that I didn't see it). The impurity must contain some aromatic rings, which stick very hard to the graphite.

Quite odd problem - either no retention or no elution. I will try Vlad's approach now. I will let you know how it goes when I have recieved the column.

[yaakov]

Mattias,

Did you try Anion Exclusion Chromatography?

[Bryan Evans]

(sorry - my last post on this discussion since it is not related to original post)

At neutral pH both sulfonic acid groups are going to be fully ionized and unless Scherzo SM-C18 has basic group with pKa of 5 or less (modified pyridine), there is absolutely no way to elute disulfate from this column at reasonable for LC/MS buffer concentrations (less than 50 mmol). Also, I don't think that buffering capacity of ammonium formate is good at neutral pH (6-7)

- Sorry, but no information has been released as to the identity of ionic ligands on Scherzo SM-C18.
- Correct. A base without it's conjugate acid is not a true buffer solution. It's a salt solution.
(though some may argue that it still resists change in pH more so than pure water).

Either way - it is useful for IEX.