Chromatography Forum

Our lab does routine analysis of sugar nucleotides, primarily using ion-paring reversed phase methods. Although this works, it's less than ideal, especially for LC-MS analysis.

Recently hydrophilic interaction chromotography has been suggested to us for use in separating the highly polar NTPs, NDPs, NMPs, & NDP-sugars which we work with. Does anyone have any experience with using HILIC in separating nucleotide-based compounds? From my review of product brochures and literature, it seems that HILIC is aimed more toward resolving basic compounds, versus acidic phosphates.

Thanks.

I have developed a method for nucleic acid bases separation by HILIC. But sugar and phosphate were no longer present and we separated only purine and pyrimidine bases adenine, thymine, cytosine, guanine and uracil. I don’t know if it would work with a sugar based nucleotide, but I used a Luna aminopropyl column from Phenomenex with
A: Acetonitrile
B: HCOONH4, 10mM pH3,0

With an injection in 100% A followed by an elution by a shallow gradient of B.

Have you thought trying a reversed phase column with increased polar retention (low carbon load, polar embedded group...)? That could allow removing of ion pairing reagent and give you MS compatibility.

Hope this help