I am interested in separating two small organic molecules by ion exchange chromatography.
Following the example set by several old papers for these molecules, I want to use a cation exchanger. Both molecules have guanidino groups which bind very well to the resin. Older papers used dilute ammonia to neutralize the guanidinos and elute the molecules but I found a rather poor separation with this approach.
There is a range of pH where both molecules would have different net charges, but in that range of conditions, the guanidinos would still be charged on both molecules, and the net charge changes are different mostly because of the number of negatively charges group elsewhere on the molecules (which, in either state, charged vs. uncharged, are not expected to interact much with the resin).
My question is the following: for predicting whether a molecule is expected to elute, what matters more: the overall net charge, or the "local" presence of the ad hoc resin-binding group?