Can't seem to wash out analyte - BEH Amide
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.I'll try to give pertinent details in case these help:Acquity-Quattro MicroBEH Amide column (2.1x50)5 uL Full Loop modeFlow 0.4mL/minA=0.1%FA in H2O; B=0.1%FA in ACNGradient: 60%B to 30%B in 1min (hold 1min) then back to 60%B.Cycle time allows for ~4 column volumes at initial conditions before next injectionWNW(use 500uL)=75:25:0.1 ACN:H2O:FASNW(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)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.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).Any thoughts?!?