Chromatography Forum

Why not salt gradient instead of relatively extreme pH gradient, which degrates the compound?

Best Regards

HW> I haven't tested pH 7 on the Oasis-WAX. Waters instructs to use 5% NH4OH for elution. That is anyway way too basic for my molecule, maybe pH 7 is perfectly OK.

One good thing about using the strong acid for SPE elution is that I can clean the cartridge with 50/50 formic acid and MeOH and wash out almost everything else but my impurity (this is only one impurity out of many in the sample). Only my "sulfonic acid" stays. I have now reached 98 area% pure impurity solutions, just by doing SPE. No need for fraction collection. If I had used base for elution, every anion in my sample would be eluted together.

Regarding the LC-method, please give suggestions of other LC-columns (brand, name etc) that could do the same "trick". I guess they must be out there, but I have no idea where they can be found. Waters and Agilent only have SAX columns in their cataloges. They contain quarternary amines that will never be "shut off".

Danko> Want to run LC/MS...

You know as well as I do that each type of column has its own characteristics, but I don´t know of any manufacturer of ion exchange columns who does not have the weak variety, which can ALL be shut off.

In most cases people call "weak" group if it is primamry, secondary or tertiary amine with pKa 9-11 and call quaternary amine as a "strong" anion-exchange. You need to go to a higher pH (10-12) to shut down anion-exchange properties on WAX and it is almost impossible to shut down quat. In each case you need to consider stability of the analyte at high pH and in case of Mattias it is not stable at higher pH. Even if we assume that compound is stable at higher pH, if you go higher in pH all acidic fragments will be ionized and molecule will become more acidic.
What else might work here is pyridine-based column, which you can gradually "shut down" by going to pH 5.5-7 (without going to pH extremes)....but why anybody wants to buy a new column, do method development to if SIELC already developed a method?

Just trying to eliminate some myths, like: "if you go higher in pH all acidic fragments will be ionized and molecule will become more acidic."
Nobody suggested a new column, but it would be interesting to see what other columns, not tried here, or other conditions could do.

I have just hooked up the Obelisc method to the QTOF and, as expected, the unknown molecule has excellent response in negative mode. The MS background using weak NH4OAc and acetonitrile was close to nothing.

The mass and isotopic pattern was determined using lock mass infusion, and all top 20 hits of possible molecular formulas show 4 or 5 sulphur atoms. Time for collection of sample for NMR...

Mattias -

Thank you for sharing all your data and results. As you said, doing this without (volatile) ion-pairing for MS analysis is a real challenge.

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Vlad, it appears that this is a combination of RP + IEX. And that the IEX was shut down by doing a pH gradient from 5-7, correct?
It's confusing because this is supposed to be a NP column, and yet the data you show (40% acetonitrile) looks to be RP?
Do we know what world we're in?

Obelisc N is HILIC column with two ionizable groups (cation- and anion-exchange) in this particular case column is used in cation and anion-exchange mode with adjustment of column strength by pH gradient