acidic and basic analytes...which column?

<p>Trying to develop UPLC-MS (ESI) method for anlaysis of an aminoglycoside (very polar and very basic!) and a glycopeptide (also polar but less basic).  I can retain the GP on HSS T3, but not sure where the AG is.  Oddly I thought I had it down when using 0.1%FA in H2O with shallow gradient using 0.1%FA in MeOH.  Actually saw two peaks so thought I was golden.  30 or so injections later, can't see the AG anymore!?!?  Where oh where did it go?  I've thought about HILIC and have the Amide column to try but the thought of switching everything out kind of bums me out.  Besides not sure what'll happen to the GP.  Any thoughts from the better chromatographers than I that I know are out there?  Thanks!</p>


  • Evelyn


    You may wish to try ACQUITY HSS PFP column, with ion pairing.

    HILIC works for some, but not all AGs, and you get broader peaks.

    Hope this helps.

  • dougm

    Do you have a literature reference that describes a successful separation of the same (or similar) analytes? That may help us make a better suggestion (or two).

    HILIC uses the same mobile phase(s) that you use now but uses them in reverse. Water (or 0.1% HCOOH) is your strong eluting solvent and ACN is the weak eluting solvent. No need to change anything. HILIC is a powerful tool that can solve some chromatographic problems that you just can't do using reversed-phase LC. Think saccharides/carbohydrates. No way you can retain and separate such compounds using reversed-phase LC. No need to get bummed out about HILIC - just take a look here.

    Regarding your other post about the HSS T3 chemistry. The HSS T3 column is medium ligand density C18 column that you can use with mobile phases containing as little as 0% organic modifier. (or 100% aqueous). So, you can treat it just like any other C18 chemistry. No special care, or different care, is required. As Beowulf mentioned, the Care & Use Guides (HPLC & UPLC columns) do a pretty thorough job of describing the basic care and feeding of the HSS T3 chemitry: link.