regeneration/cleaning biobasic AX column

[Bas251087]

Hello,

I am working on a method for the determination of phosphate compounds in whole blood with LC-MS.
For the separation, I am using a biobasic AX (weak anion exchange) column from Thermo Scientific. But ~800 injections resulting in broad peaks and bad peak shapes.

The conditions are:

Biobasic AX column: 50x2.1 mm 5 um
Injection volume: 5 ul
Column temp: 50 degree celcius
Flow: 0.25 ml/min
mobile phase A: 10/90 (v/v%) acn/h20 + 10 mM ammonium acetate pH 6
mobile phase B: 10/90 (v/v%) acn/h20 + 1 mM ammonium acetate pH 10.5

Compounds are trapped with pH 6 and eluted with pH 10.5. A higher conc. of acn resulting in peak tailing.
A lower column temperature resulting in peak fronting.



1. Proteins from whole blood samples are precipitated with 10% trichloroacetic acid
2. The supernatant is extracted with tert. methyl butyl ether and the aqueous layer is injected. The addition of LLE from step 2 resulting in less matrix effect.

After each batch the column is flushed with a gradient of 10% acn to 90% acn without salts.

Does anyone have experience with regeneration/cleaning this kind of columns?

[tom jupille]

It may not be worth it. 800 injections per columns is not too bad for bioanalytical work. Look at it this way, it's about $0.50 per injection. I suspect you're spending more than that in paperwork time.