Chromatography Forum

hi

what does Ion capacity or Ion Exchange Capacity stands for in an Ion Exchnage column?? (eg.. Dionex DNApac column shows anion exchnage capacity of 40 meq/column)

how does this values effect my column selection and method development??

Hi Rick,

It tells you what the sample load capacity is. The larger the number the higher load the column can take.
Keep in mind that it’s not the mass but the ionized/interacting sites of the analyte (under the given conditions) that is relevant in this context.

Best Regards

To clarify then,

40 meq = 40 milliequivalents ≡ 40 mmol analyte with 1 charge site?

Is it really independent of ion/molecular size?

ion exchange capacity is the concentration of charge per unit column volume (usually). While, it does affect loading capacity, it has other, more important implications. The concept is roughly comparable to that of specific surface (m²/g) in a reversed-phase column; both represent the phase ratio (amount of stationary phase available). In both cases a column with a higher capacity (or surface) will be more retentive and require a stronger mobile phase for elution.

It's probably more important in ion-exchange because there is a tremedous range of availalble columns. Polystyrene sulfonate cation exchangers that I've worked with in the past, for example, can range from 10 μeq/mL to 4 meq/mL. A very weakly bound in might never be retained on a low-capacity column, while a very strongly bound ion might be impossible to elute from the high-capacity column.

40 meq = 40 milliequivalents ≡ 40 mmol analyte with 1 charge site?

The rationale is correct, but if the value/unit is per column, then 40 mmol analyte with 1 charge site, would be huge overload, because you don’t want the analyte to be distributed through the whole column, but hold it within a discrete (and hopefully) narrow band.

Is it really independent of ion/molecular size?

The load capacity is (more or less). But when it comes to retentivity and selectivity then the answer is: No.
Both the ion/molecular size and conformation affect these two factors greatly.

Actually, if the column is not overloaded and is used under standard elution conditions (gradient protocol), the retentivity is not that hugely affected by the capacity, unless the gradient is very, very shallow. The strong eluent (when initiated) will typically contain many, many more ions than there are counter ions bonded on the column material. So, when the strong eluent arrives (in the required concentration), the analyte is kind of “free to goâ€

retentivity is not that hugely affected by the capacity, unless the gradient is very, very shallow.

I'm not sure whether I agree with that. I do agree with the rest of your post, but any given analyte will elute later (i.e., at a higher ionic strength) from a higher-capacity column.

Hi Tom,

I do agree with this analogy and I would expect that it could be proven - provided the gradient is very shallow, as I tried to emphasize in my previous post. In that case the analyte would be attracted to somewhat larger number active sites on the stationary phase and thus get retained for a longer time.
But the analogy is much more valid in reversed phase mode than in ion exchange mode in which the attraction and the following release of the analyte has more of a digital (yes/no) behavior. So, if the analyte is released from one active site I wouldn’t expect it to be attracted to the next one in the immediate “neighborhoodâ€

Danko: your argument makes, sense, but I think it hinges on two assumptions which do not generally apply:

A). An on-off mechanism, which isn't really the general case (and only a simplifying assumption for highly-charged analytes).

B) Relatively close exchange capacities (not orders of magnitude difference) in the two cases.

Perhaps a concrete example will help (granted, it's isocratic):


The difference in mobile phase ionic strength roughly parallels the difference in column capacity (a factor of several hundred).

Hi Tom,

Thanks for illustration. It would’ve been better yet, if everything else was kept equal (i.e. mobile phase chemical composition, concentration, pH, etc.). Still, I predicted a similar outcome provided the gradient was very, very shallow (can’t get shallower than isocratic)
The digital (on/off) behavior on the other hand is observed in gradient mode (as I emphasized) which is by far the most beneficial application of the ion exchange technique - In my world anyway (protein separation).

Best Regards

Tom,
I’ve just noticed – on second review. The titles of the two chroms you’ve posted are mismatched. The 4 meq/mL should refer to the “low capacityâ€

[quote="danko"]Tom,
I’ve just noticed – on second review. The titles of the two chroms you’ve posted are mismatched. The 4 meq/mL should refer to the “low capacityâ€

Maybe it’s time for me to make an appointment with the local optician

Best Regards

I'll have a go at the mental experiment:

For a column of given capacity I would expect the same retention time for both the 2 mmol and 4 mmol sample load assuming that the latter does not cause 'mass overload' (charge site overload or whatever the equivalent nomenclature is in IC.)

However, I feel this example is distinctly different from the case where load is kept constant and capacity is changed. As capacity refers to the density of retentive sites on the sorbent I visualise this simply as the distance the ion or analyte travels before being re- ad/absorbed. Analogously, reversed-phase retention will increase on a C18 with increased bonding density.

I’m beginning too realize (more and more) that we are in agreement with each other.
So, would it be fair to summarize it as follows?
In isocratic mode (and even with very, very shallow gradients) and with small molecules the charge density will affect the retention time significantly. However, with larger molecules, such as proteins, and standard (steeper) gradients, the retentivity would not be influenced to the same degree (if at all) because the counter ions in the strong eluent will “meetâ€

Ion exchange of proteins is not an on-off mechanism as the reversed-phase chromatography of proteins is. One can actually run quite comfortably isocratic ion-exchange of large molecules. This is very difficult with reversed phase.

The basic reason is that the hydrophobic interaction area responsible for retention in RP increases with molecular weight. In ion-exchange on the other hand, one can conveniently manipulate the charge of a large molecule by changing the pH. Thus you can make even large proteins run with 1 to 3 charged groups, and they do not behave any differently than small ions.

Other more subtle things play a role as well...