Can't seem to wash out analyte - BEH Amide

<p>I am having trouble washing out a very polar analyte from my BEH Amide (HILIC) column.  Sounds funny as I'm writing this, because what could be easier!?!  But alas, I've tried all kinds of things with no luck.</p><p></p><p>I'll try to give pertinent details in case these help:</p><p>Acquity-Quattro Micro</p><p>BEH Amide column (2.1x50)</p><p>5 uL Full Loop mode</p><p>Flow 0.4mL/min</p><p>A=0.1%FA in H2O; B=0.1%FA in ACN</p><p>Gradient:  60%B to 30%B in 1min (hold 1min) then back to 60%B.</p><p>Cycle time allows for ~4 column volumes at initial conditions before next injection</p><p>WNW(use 500uL)=75:25:0.1 ACN:H2O:FA</p><p>SNW(use 800 - 1000uL) = variable attempts.  Have so far tried 75:25 H2O:ACN  (with and without FA---up to 0.2%) and 100% H2O (w/ and w/o 0.1%FA)</p><p></p><p>I have 2 analytes - a glycopeptide (0.7min RT) and an aminoglycoside (1.25min RT).  The AG is the problem child whereas the GP washes out even if you just ask it nicely.</p><p></p><p>I'm sure it's  a washout issue because I can run 2 consecutive blanks and the AG area decreases from 1st to 2nd blank (whereas GP is baseline even in 1st blank).  I think this problem is the root of my horrible peak area RSD's for the AG - can only get  >5-10% RSD (whereas GP consistently gives ~1% RSD).</p><p></p><p>Any thoughts?!?</p>


  • Have you considered that you may be experiencing injector carryover ?

    This seems to be the simplest explanation.

    Managing carryover is a matter of selecting appropriate Sample Manager wash solvents to rinse away sample components that may adsorb to various surfaces.

    It sounds simple but it can be challenging.  There is as much art as there is science in the solvent selection but you might consider something that is more rich in organic solvent.  For example, 50% water:25% acetonitrile:25% isopropanol.

    The latest version of the ACQUITY Instrument Control Software & Firmware (June 2011 Driver Pack, version 1.5 software) has added a function to help manage carryover. It allows you to rotate the injection valve during the strong solvent flush portion of the gradient.   This has proven effective at dramatically reducing injector carryover

    This new software also gives you a tool to help diagnose whether the carryover is coming from the injector or the column.  It allows you to run a gradient without making an injection.  This is different than simply making a 0 (zero) uL injection.  0 uL injections still result in the rotation of the injection valve whereas the No Injection function does not.  So, if a No Injection run results in a peak, it is reasonable to assume that the carryover originates in the column and not the injector.  Some tips for managing column carryover include reducing the slope of the gradient (more gradual changes in composition) and increasing the time of the strong solvent flush step.

    Contact your local Waters Representatives for additional details on obtaining the latest software.



  • <<Repeatedly slaps self on forehead>>

    I agree it is most likely injector and/or needle carryover and believe it or not that is what I had in mind, but for whatever reason I wrote my post in terms of the column.

    I am very intrigued by this new software version and will check with Waters on how to obtain that.

    Rich, you suggested more you mean in the SNW?   I'm trying to be mindful of H2O being the stronger solvent in HILIC.

  • Yes, I am referring to the SNW.

    I have no compelling reason to think it might help other than the recipe used for the attached application note for veterinary antibiotics (including some aminoglycosidic ones).

    My standard needle wash is what I call Wonder Wash.  Equal parts water, methanol, acetonitrile, & isopropanol with 0.2 % formic acid.

    It generally works well but there is always that stubborn compound that requires the skills of an alchemist !

    Good luck,


  • Hi

    I assume you are using the standard PEEK needle in the sample manager, so another option is to try the SS needle. We have seen differences in carryover between these two needle types, so might be worth trying if a change in wash solvent does not solve the problem.

    best regards


  • Well actually.....I am currently using SS.  Perhaps a switch to PEEK could be tried.  As it stands I'm still in the market for a solution to this problem.  I tried the Draino (25:25:25:25:0.2) with no luck.  In fact my blank actually grew!  Oooops.  So i'm back to 75:25:0.1 H2O:ACN:FA which so far gives the least, worst results (if that makes sense!), but still gives uncomfortably large blanks.  Grasping at straws here, but does the Seal Wash matter when trying to solve carryover issues?  I'm currently running pure MeOH (for no good reason other than i've heard MeOH "wets" things fairly nicely).  Does the fact the I have no MeOH anywhere else in my system matter?

    On a somewhat related subject, all this switching about of wash solvents makes me remember that you're supposed to characterize seals, loop volume, etc. when you change stuff out.  Does anyone have intimate experience with this idea and can tell me how significant this is to do?  If you don't do this, what does it affect?  What all needs to be re-characterized?  Is this idea only important if you change mobile phase...or do wash solvents count as well?

    Thanks again for any advice you care to offer!

  • Hi again

    Yes I would try switching back to the PEEK needle to see if there is a difference. The seal wash does not come in contact with the sample or injector, so it should not matter. In general seal wash should be more aqueous since this is normally the best to dissolve buffer salts, so I usually use 10-20% organic in water.

    When you change the composition of the weak wash solution you should characterize the needle and loop volumes. Since the weak wash is in the needle when the sample is drawn its composition will affect the positioning of the sample. If not characterized properly it could effect precision and reproducibility of the injection, but not likely the carryover.

    Have you tried to switch from full loop injection mode to partial loop to see if this affects carryover ?  In full loop mode with a 5 ul loop the overfill factor is 5 so 25ul sample is drawn, and the extra 20ul needs to be flushed from the injections system after the injection which might be the thing leading to the carryover you see. In partial loop mode (not PLUNO) only the injection volume is drawn.

    best regards


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