Chromatography Forum

Hello,

we currently try to set up a method for Paraquat/Diquat with LC-MSMS on a zwitterionic HILIC column but have trouble to get them eluted from the column. We use 20mMol Ammoniumformate pH=3 and Acetonitril as mobile phases. We tried columns from different suppliers (Sequant ZicHILIC and MN Nucleodur HILIC) but we have to lower the percentage of ACN down to 20% to get them properly eluted from the columns. From the available applications they should be eluted much easier.

HPLC-System: Agilent 1290

Has anyone observed the same problem and any idea why we observe this phenomenon and why and where stick on the column?

Best
Axel

There is a validated method for paraquat and diquat on our Obelisc R mixed-mode column which is currently running in about 80 organizations worldwide.

Here is a reference manual (validated in Europe): http://www.crl-pesticides.eu/library/do ... _QuPPe.pdf

I think you might want something less polar for your stationary phase if you're having such a hard time. I'd also suggest a milder pH (4-4.5) for your buffer.

I've used Waters' Amide BEH to separate polysaccharides, and it worked beautifully. Being that you've got a charged analyte, I'm not sure if its the right choice, but its a start. A Zwitterionic column is overkill polarity for your analytes, and may not work at all to do what you want.

The first problem I can see is that the stationary phase is charged, as are your analytes. The analytes are almost definitely adhering to your column electrostatically, and unless you're using a seriously salty buffer (read: instrument damage) you're not going break that interaction terribly easy without a high percentage of water.

Do you have a 1290 binary or 1290 quaternary pump?