Anyone ever used BEH-Amide for NDP-Sugars?

Hope someone can help me out.  I have a user who is trying to finish  up his degree and leave, but needs me to do LCMS on some NDP-Sugars he  has synthesized.  He doesn't have time to purify them and his LC method  isn't MS compatible.  I'm trying to use a BEH-Amide column, but don't  have much experience with this chemistry.  I've been using a gradient  method (5 min 85-70%B, hold @ 70 for 5 min then back to 85 and hold)  using acetonitrile for B and 5 mM ammonium acetate for A.  If I do a  long pre-equilibration I get retention, but in subsequent injections I  don't get retention.  I'm hoping someone has some tips to help me out.Thanks,Cameron

Answers

  • Hi Cameron,

    I haven't used BEH Amide for NDP Sugars, but it sounds as though you have developed a method that does retain and separate them. You need to define the size of the column, the flow rate, and what the injection to injection run time is. You say retention is present when the column is pre-equilibrated for a long time. How long is a long time?

    The attached 'Efficient Hydrophilic Interaction Chromatography Method Development' wall chart suggests that 5 to 8 column volumes worth of re-equilibration wll be required between injections. Build that into the gradient method. This minimum re-equilibration time will need to be observed before every injection. You can use the Acquity Column Calculator program to generate column volume information, which will depend of course on the column dimensions and flow rate.

    Ken Blakeslee

  • Ken,

    Thanks for the pdf. I think my problem is the changing ionic strength,

    since my AcN has no modifier. I'm going to try making new A and B

    solvents both with 5-10 mM ammonium acetate. I'm running a 2.1 x 100 mm

    at 0.3 ml/min and using a post gradient re-equilibration of 10 min.

    I'll let you know how my runs with the new solvents work.

    Thanks again,

    Cameron

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