Chromatography Forum

Hi all,
I have worked with the amide column and after 400 injections, the column has died, so what Waters recommend is to use an amine column. Any thought?
Thank,
Carol

Without deeper knowledge of the method used and how exactly the column "died" it's hard to give a judgement.
Generally speaking, an amide column should be much more (chemically) stable than amine, therefore that recommendation seems a bit odd to me.

Hi again,

I used the column for Lactose analysis in milk the method recommended by waters with 0,2 % of TEA, I don't know why, this result in high back pressure problems. I have read similar observations and problems in the forum.
I am still using it but with acid conditions, without pressure problems, however, the retentions of the peak are not those achieved initially.
thanks,
Carol

At neutral or acidic pH, HILIC separates the alpha- and beta- anomers of reducing sugars. These are connected by a continuum which corresponds to the open chain structure. At elevated pH, the rate of mutarotation increases and the anomer peaks collapse into a singlet peak. That's why one adds 0.1% TEA or some such when using a neutral column. With a column with a basic (= (+) charged) surface, the local microenvironment has a pH high enough that TEA is not required. That's probably why you were advised to switch to an amino column.

o.k,
Very useful information!!

Thanks,
Carol

Hi all,
I have worked with the amide column and after 400 injections, the column has died, so what Waters recommend is to use an amine column. Any thought?
Thank,
Carol

Could you describe you mobile phase? The amide phase is typically more stable than an amine phase. But in general, all hydrophilic, very polar bonded phases are much less stable than the typical reversed phase chemistries. Your observation is not unreasonable.

Hi all,
I have worked with the amide column and after 400 injections, the column has died, so what Waters recommend is to use an amine column. Any thought?
Thank,
Carol

Actually I found an amine column MUCH less robust than an amide for our samples. We typically get somewhat around 1200 or more injection on the amide analyzing for mono and disaccharides from a pretty messy matrix. No where near for the amine.