HILIC - BEH Amide storage soln.

<p>I have an assay using BEH Amide (HILIC) column and fairly clean samples (salines that have been SPE'd....no proteins).  Gradient elution using mobile phase composed of various %'s of [ACN+0.1%FA] and [MilliQ+0.1%FA].  The column gets fairly constant use (at least 3 times per week).  For my shutdown method I just wash with 50:50 of MPA:MPB, so the point is that the column is ALWAYS under 0.1%FA conditions, even during "storage".  Does anyone think this may be a problem?  My RT's are rock solid day in and day out, but I am seeing some *wild* fluctuations in peak area day-to-day and am wondering if this is related in any way to storage conditions.  My thoughts were to keep the stationary phase under one consistent pH condition at all times.  But am I missing something?</p>


  • I know on some older Waters HPLC columns, storing with even small amounts of TFA messed up the column packing material. Try a mild buffer/ACN solution to store the columns, without any harsh organic acids. What exactly is in your mobile phase? I've run some methods that TFA (even at 0.05%) chewed up the packing material after 80-100 injections (Agilent 1200 LC+Waters column run by Empower2, and tested on an Alliance LC with the same result). TFA is generally not kind to the packing material, in any regard.

    (Side note: are you actually disengaging the column from the instrument, or are you leaving it on the instrument under low flow?)

  • In general, for periods longer than 4 days, the recommended storage solution is 100% acetonitrile. The flushing and storage procedure is described in the ACQUITY UPLC BEH Columns Care & Use Guide. However, as you've discovered, there is little harm storing your column in your mobile phase for short periods of time in order to reduce column equilibration upon re-installation.

    I'm not sure what would cause wild fluctuations in peak area but I am pretty confident that it is not your column storage conditions (which look fine to me).