Enantiomer separation on an achiral polar embedded column

[kjwellsknecht]

I've been observing the separation of N-oxide enantiomers on a Varian Polaris column and wondering if anyone else has observed chiral separations on an "achiral column". I don't have definitive proof that the two N-oxides are enantiomers but they have the exact MS/MS spectrum. No special mobile phases involved, just ammonium formate (pH 3.5) and ACN. The are definitely N-oxides confirmed by 2H-exchange experiments. There is only one nitrogen available for oxidation with MCPBA. I don't know how else to explain the two peaks.

[Mattias]

It is quite common to have some separation of enantiomers in reversed-phase without any special columns or additives. But I guess it can just as well be a problem with chromatography in this case.

A quick check could be to lower the injection volume to a minimum and see if the peaks merge to one peak (=chromatography problem). Then you might just be injecting to much, or having too strong sample solvent. Have you tested it on a brand new column?

[HW Mueller]

Are we talking about optical antipodes? Separating these without chiral interaction??
I would have taken two peaks, if they are really two substances, as proof that they are not "optical isomers".

[kjwellsknecht]

N-oxides can exist as R or S isomers so yes, they are mirror images of each other or enantiomers. Technically, they should have identical physical properties so one would expect them to coelute on an achiral column. However, I'm not sure if when you use a C18 column with a polar embedded group if multiple interactions can occur which would allow some chiral separation. No effect when I try different injection volumes. Not sure if I've tried a new column but that's a good suggestion. I'm typically analyzing biological samples (microsome incubations, plasma extracts, etc.) so perhaps small amounts of protein/peptides are bound to the column that could have interactions with the enantiomers and cause some chiral separation.

[lmh]

Absolutely, HW Mueller. There are plenty of structural isomers that are not enantiomers and nevertheless have identical MS2 spectra (within experimental reliability), but it's hard to think of any theoretical situation where enantiomers would separate on a column that is genuinely free of any chiral features. Of course gunk stuck to the column may be chiral and may interact with an analyte.

[Alex Buske]

Hi,

Could the n-oxides be diastereomers? Or is the other part of the molecule symmetrically? As soon as the different orientation at the nitroge leeds to different molekule shape separation on achiral columns is possible.
Alex

[DM909]

I am not aware that you can have enantiomeric separation on a non-chiral RP column. Identical MS is not a guarantee that your molecules are identical either. Unless they are diastereomers as Alex suggests, I really don't think they're separable.

To me this sounds like a chromatography artefact - split peak. That would also explain the identical MS. Is your sample matrix very different from the mobile phase? Is your retention time low? If these two are yes, then you can have peak splitting. Also, if your concentration is too high your column can be overloaded and you'll also get peak deformities which can appear as two peaks. Do your peaks have good shape? Do they have front tailing? If yes, then there's a good possibility that your concentrations are too high.

[Mattias]

I guess I was misusing the word enantiomer when I wrote that "it is common to have separation...".

I was thinking of separation of peptides where one amino acid is in D-form instead of L-form - which is no problem at all on RP-columns. But I guess they only qualifies as stereoisomers - not enantiomers.