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- Posts: 252
- Joined: Sat Nov 07, 2009 6:27 pm
I am interested in separating two basic peptides on WCX. These peptides are >95% identical. One has 10 Args while the other has 9.
These peptides elute when the salt in the mobile phase is around 250 nM.
In order to increase resolution, gradient time is increased or, the increase in [salt] is made flatter (for instance, running a gradient from 200-300 mM
My question is whether the peptides ought to be loaded under low salt conditions (10 mM), followed by a 5 min ramp up to 200 mM (then 60 min grad to 300 mM) Or, if I can simply equilibrate the column at 200 mM salt and start directly at this higher salt concentration.
There are instances when I may have to load a large sample volume (several mL on a 4.6 mm column). I'm concerned that, although the peptides may not elute at 200 mM salt, that loading under these high salt conditions may lead to broadening.
These peptides elute when the salt in the mobile phase is around 250 nM.
In order to increase resolution, gradient time is increased or, the increase in [salt] is made flatter (for instance, running a gradient from 200-300 mM
My question is whether the peptides ought to be loaded under low salt conditions (10 mM), followed by a 5 min ramp up to 200 mM (then 60 min grad to 300 mM) Or, if I can simply equilibrate the column at 200 mM salt and start directly at this higher salt concentration.
There are instances when I may have to load a large sample volume (several mL on a 4.6 mm column). I'm concerned that, although the peptides may not elute at 200 mM salt, that loading under these high salt conditions may lead to broadening.
