Ion exchange SPE

[JA]

I'm looking at the clean up of a sample containing a polyamino-functional cpd, a short alkyl sulfonate (sodium mercaptoethane sulfonate) and a currently unidentified cpd, suspected product of the reaction of the previous two. On a YMC Hydrosphere C18 running 98:2 v/v H2O+0.1%HCOOH / MeOH+0.1%HCOOH isocratically these three elute with k ~ 0.5, 4 & 6 respectively.

The supplied sample is a crude reaction mixture in glacial acetic acid, also containing an excess of sodium acetate.

Ideally I would like to enrich a sample of the unidentified product for further characterisation however I am struggling to retain it by SPE. I tried, and ruled out straight reversed phase SPE due to the apparently similar hydrophobicity of the product and alkyl sulfonate. I suspect that I can fractionally elute the product from the polyamino starter if only I can firstly do away with the alkyl sulfonate.

I cannot seem to retain the product on a strong cation exchange phase. I've tried by using the crude, diluted rxn mixture, and also by isolating the rxn mixture components by evaporating off the acetic acid and then freeze-drying. Question: is either the rotary evaporation (under reduced pressure) or freeze-drying step likely to remove sodium acetate? I'm wondering if this, or the alkyl sulfonate sodium counterion is interfering with the SPE loading step?..

I've tried loading using a 40 mg/ml soln of freeze-dried rxn mix in 0.1% H3PO4 (pH ~ 2.2). Loaded 1 ml of it on a 500 mg (6 ml) cartridge. Is it advisable to load a weaker sample using higher volumes?

Any advice appreciated.

[HW Mueller]

How about using a basic mobile phase to retain the amine only, then, if needed, an acid phase to get the amine out (or organic?).

[Uwe Neue]

Since your sample is protonated already, I would not add any phosphoric acid. If you have more of the freeze-dried sample, I would dissolve it in water, test the pH add a bit of acetic acid to get it to a mildly acidic pH (5 is possibly enough) and then retain the polyamine compound on the strong cation exchange SPE. The mercaptosulfonate would brake through, and your unknow compound will either do the same, if it is not an amine, or it will stay with the amine. If it is what you think it is, you may be able to fractionate it from the amine by manipulating the elution pH.

[JA]

HW Mueller,
Thanks, I'd completely missed trying high pH on the reversed phase cartridge. I'll see how that goes.

Uwe,
The rxn mix is ~ 80 mg/ml total crude in glacial acetic. Originally I had just diluted it by ½, giving a sample at pH ~ 1.9 and loaded 1 ml on a water-equilibrated 500 mg cartridge. I worried that the acetic acid was causing breakthrough (can it even act as an eluent when mixed with water?). Since then, I am making samples from the freeze-dried rxn mix at 40 mg/ml in 0.1% H3PO4, pH ~ 2.2, again only loading 1 ml. Can this low volume of concentrated sample pose a problem vs. for example, loading 10 ml of 4 mg/ml sample (pH ~ 2.4)?

The phase by the way, is Strata-X-C. All we had in the lab

After this, I am having trouble visualising how your proposal is significantly different from what I have tried. Even at those low pH's the polyamino is not sticking

[Uwe Neue]

The sample is highly concentrated, but this is not a principle problem unless the sample is very viscous. In principle, one should be able to load 80 mg of sample on a 500 mg bed, but you must realize that you are pushing it. Since you are having trouble, I would go to a lower amount (you did this with the 40 mg already, but maybe this is still too much, and all you can load with your sample is 20 mg). The reason that I recommended a lower pH is that with the phosphoric acid, you are in a strongly acidic regime already, and there is no reason to be there. Don't forget that one can regenerate such packings by washing them with a strong acid.
Try the pH 5-ish, maybe combined with a smaller load.