Chromatography Forum

Did anyone work with NH2 columns on normal phase? What is a typical gradient for NH2 column on NP?

I have problems with a product that break on RP. On NP using silica, the product was retained on the column.

Thanks in advanced

Than I would highly recommend a Standard HILIC column, that will solve your Problem.

Yes, an amino column can be used in "traditional" normal-phase mode. A typical starting point would be similar to that for silica: a "scouting gradient" starting with 1% isopropanol in hexane and running up to 100% isopropanol. That's more or less the equivalent to the standard 5% - 100% ACN scouting gradient in reversed-phase. Note that the relationship between mobile phase strength and composition in normal-phase is non-linear, so that a disproportionate part of the strength increase occurs in the first 10% of the gradient (but that's true for silica as well).

That said, if your compounds are very polar, and especially if they have ionizable functional groups, then Gerhard's suggestion about HILIC is a good one.

Gerhard, Tom

thank you very much with your solutions.

My first option for NH2 column was HILIC, but it is a non polar compound. That the reason to suggested NP.

Tom, I would try to use Heptane - EthylAcetate in a gradient, what do you think about it? I have never used Isopropanol in our lab on NP.

Thanks in advance

Tom, I would try to use Heptane - EthylAcetate in a gradient, what do you think about it? I have never used Isopropanol in our lab on NP.

No problem. The reason for suggesting IPA is that it is the most polar solvent that is still miscible with hexane (not sure about heptane) and it has a low UV cutoff. EtAc is fine, it just gives you a bit narrower polarity range and a higher UV cutoff.

Thanks Tom

I'll tell you what happen

i'm puzzled: if you're not using reverse phase because your compound isn't stable in reverse phase conditions, why would it survive hilic any better anyway? If you're working on an analyte that degrades rapidly in water/acetonitrile, then it's likely to be a struggle to handle it...

Here is our alternative approach for analysis of polar molecules in hydrogen-bonding mode - SHARC 1 column (Specific Hydrogen-bond Adsorption Resolution Chromatography)

http://www.sielc.com/upload/file/pdf/SHARC_1.pdf
For molecules which are polar and which are not stable in aqueous mobile phase