Chromatography Forum

Hello Everyone,

Recently we bought Atlantis, HILIC OBD prep 5 µm, Dimesion 19 mm× 150mm column for the purification of hydrophilic compounds.

The mobile phase mostly used in HILIC are 90/10 acetonitrile/ 100mM ammonium formate pH3.

My question is it necessary to use buffer ammonium formate or acetate to retain hydrophilic compounds, as my compound are sensitive to low pH.

Can I used only water and Acetonitrile to achieve good separation?
any suggestion will be highly appreciated.

Regards.

If you compound is neutral or surface of silica does not have ionizable groups of opposite charge to your analyte you can avoid buffer. If your compound has ionizable groups which can interact with stationary phase by ion-exchange mechanism then you need ions in the mobile phase to facilitate elution.

Basic analytes are the most polar (or at least the best-retained in HILIC). This widespread use of buffers with pH of 3 or so reflects applications involving peptides. Below pH 4, peptides lose the (-) charge on carboxyl- groups. This leaves them with a net (+) charge. They are retained most strongly under those conditions. If your analyte of interest doesn't have any carboxyl- groups or if increasing retention time isn't a concern (i.e., being able to get adequate retention at a lower % ACN), then there's no need to use so low a pH. Use ammonium formate or acetate right out of the bottle if you like (with resulting pH's around 5.5 and 6.5, respectively). I do recommend having some salt present, if only to insure that there's a reproducible layer of counterions on top of the (presumably somewhat charged) stationary phase surface. That will affect reproducibility. Try it with and without 20 mM salt (that's 20 mM overall, not just in the aqueous portion of the mobile phase).

If you compound is neutral or surface of silica does not have ionizable groups of opposite charge to your analyte you can avoid buffer. If your compound has ionizable groups which can interact with stationary phase by ion-exchange mechanism then you need ions in the mobile phase to facilitate elution.

Hello Everyone,

Recently we bought Atlantis, HILIC OBD prep 5 µm, Dimesion 19 mm× 150mm column for the purification of hydrophilic compounds.

The mobile phase mostly used in HILIC are 90/10 acetonitrile/ 100mM ammonium formate pH3.

My question is it necessary to use buffer ammonium formate or acetate to retain hydrophilic compounds, as my compound are sensitive to low pH.

Can I used only water and Acetonitrile to achieve good separation?
any suggestion will be highly appreciated.

Regards.

Thanks you for the explanations. It's really helpful

Basic analytes are the most polar (or at least the best-retained in HILIC). This widespread use of buffers with pH of 3 or so reflects applications involving peptides. Below pH 4, peptides lose the (-) charge on carboxyl- groups. This leaves them with a net (+) charge. They are retained most strongly under those conditions. If your analyte of interest doesn't have any carboxyl- groups or if increasing retention time isn't a concern (i.e., being able to get adequate retention at a lower % ACN), then there's no need to use so low a pH. Use ammonium formate or acetate right out of the bottle if you like (with resulting pH's around 5.5 and 6.5, respectively). I do recommend having some salt present, if only to insure that there's a reproducible layer of counterions on top of the (presumably somewhat charged) stationary phase surface. That will affect reproducibility. Try it with and without 20 mM salt (that's 20 mM overall, not just in the aqueous portion of the mobile phase).

Thanks you for the explanation.