Chromatography Forum

Hello.

We are using LC to separate nucleotides (AMP, ADP, ATP) for detection with MS.
We use Hypercarb (PGC) column, 150x2.1 mm, 5µm particle size.
Our solvents are:
A: 50mM NH4Ac + 0.1% monoethanolamine
B: 70% Acetonitrile + 50mM NH4Ac + 0.1% monoethanolamine
Gradient is 5-50%B in 10 minutes.

Using this configuration, we can detect nice peaks of nucleotides, but ADP and ATP elute at almost same retention time.
Can anybody suggest a way how to modify the configuration to separate these two compounds?

Thank you.

Ttomas

Hypercarb is rather different from most reversed-phase packings in that a great deal of its selectivity is due to the rigid nature of the surface. That said, the variables you have to play with are:
- temperature
- gradient steepness
- organic solvent
- pH
- additive concentration.

For each parameter, set up one experiment where you make a big change in only that parameter (e.g., 15 degree change in temperature, then double the gradient time, then change from ACN to MeOH, . . . etc.) and see what happens. If you see a change in selectivity, then you can spend the time to optimize. If no change in selectivity, you will have to try with a different stationary phase.

More on pH: Hypercarb bear positive ions at some pH, which you may use to advantage. (Have some data on this, takes too much time right now to extract, but this is well known. Maybe someone can chip in with a pH range?)

If you consider an alternative, HILIC is an option.

http://www.sequant.com/sn/ufiles/SeQuan ... 00-03A.pdf

The application shows isocratic separation using a ZIC®-pHILIC column at pH 8.8, however slightly acidic conditions in fact increases the resolution ATP/AMP.