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?


1 Answer 1


Ion chromatography or ion-exchange chromatography (sadly) does not totally rely on net/local charge. Imagine the separation of standard simple inorganic ions such as fluoride, chloride, bromide and nitrate on ion-exchange resins. All of them have a single minus charge yet they have significant retention time difference as fluoride, elutes first, always, and nitrate in the end. In simple cases, one can say that the hydration radius of each ion is different and that is why they interact differently with the stationary phase (recall Coulomb's law)

The problem starts with organic ionizable molecules, here all simple predictions fail. Recall that the ion-exchange's resin's bulk is an organic polymer. You will have not only coulombic interactions, but also hydrophobic interactions. Also not all ion-exchange resins are created equal. So just saying a cation exchanger is not enough. The polymer backbone of resins interacts strongly with organic molecules. The word "poor separation" does not mean anything to a separation scientist. It can mean broad but separated peaks, or tailing or asymmetric peak shapes, or just chromatographic resolution < 1.5! You characterize chromatograms with resolution, peak efficiency, and peak asymmetry.

Coming to your secondary query whether it is the net charge or the local charge. In my opinion, it is the local charge that matters. Retention of organic compounds is not well understood. This is an example from anion-exchange resin from a nice paper (Environ. Sci. Technol. 2019, 53, 16, 9734–9743), but the same ideas apply to cation exchange resins:

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