Ion Exclusion for Small Molecule Analysis

[adam]

I've just read through the new Waters catalogue and - like others - they recommend an ion exclusion column as one way to analyze for small polar molecules like ethanol. I have to admit that I don't quite understand the logic, but would like to undersand it better.

As far as I'm aware ion exclusion columns can operate via 3 mechanisms: ion exclusion, size exclusion, and reversed phase. Now if we take ethanol, the ion exclusion mechanism doesn't really do anything to retain the molecule, and it seems to me the size exclusion mechanism also doesn't do anything to retain ethanol other than the fact that it follows a tortuous path so it come out later than larger molecules would. So it seems it is just the reversed phase mechanism at work. So, for an application like ethanol, I'm not sure why these columns are any better than a simple reversed phase column (especially one designed to retain polar analytes).

What am I missing.

[tom jupille]

Hard to tell whether an EPG column would be better or worse, but the analysis of alcohols on "ion exclusion" columns has been used successfully for almost 30 years, so it works at least pretty well .

Digging a bit deeper, the ion exclusion columns in question are usually polystyrene-divinly benzene sulfonate (essentially cation exchange resins). Unlike silica-based packings, these resins do not have pores; they are essentially homogeneous beads. These beads imbibe a certain amount of water (hydrating the sulfonates), so you can think of them as little sacs containing water at a lower concentration than the water in the mobile phase. The alcohols partition between the two phases. Because the stationary phase is less polar (less water) than the mobile phase, it *is* a
"reversed-phase" separation, but it is arguably closer to classical "liquid-liquid partition" than it is to "hydrophobic adsorption", and so not necessarily the same mechanism (yes, I'm grossly oversimplifying in both cases!).

[adam]

Tom

It's interesting; the Waters catalogue (page 138) talks about 'partition chromatography' with amino packings. Their description of that mechanism seems to match very closely with what you described above. They present ion exclusion as a separate category (page 145) but perhaps the mechanisms are not that different.

Specifically, we would be interested in analyzing ethanol in gel formulations which contain other small molecules like benzyl alcohol and transcutol as well as larger more oily stuff like triglycerides. Any ideas which would be the best way to go (we used to use GC, but I'm evaluating whether HPLC options might exist).

[Uwe Neue]

Due to the very small pore size of the ion-exclusion column, this might work for your application. Triglycerides are rather hydrophobic, but they are also larger and my be excluded from the pores and thus from interactions with the packing.

An alternative though might be HILIC. Ethanol will be retained via the standard HILIC partioning mechanism, while more hydrophobic things like triglycerides will be unretained.

[adam]

My only hesitation on HILIC is that it uses a lot of ACN, and there's currently a shortage, as you know.

Can you run an organic gradient on the ion exclusion columns - we may need that to clean the junk off.

What about the Amino partition columns (discussed on page 138). Would those be an option? Can those handle an organic gradient?

Finally, what about ion exchange. Might that work? Can that tolerate an organic gradient?

Any suggestions regarding these 3 options are appreciated.

[Uwe Neue]

None of these polymeric packings with the tiny pores is terribly good at solvent changes. Some might be a bit better than others, but unless it says it in the C/U manual that one can wash it with organic, I would not think of such an idea. On the other hand, the triglycerides that you mentioned as a problem are not expected to be retained, because they won't be able to get into the pores.

What detector will you use for the ethanol? If you are planning to use RI, then you can use acetone as the weak solvent in HILIC to replace the acetonitrile, and you can use a silica HILIC column. If you plan to use low UV, then there is no way around acetonitrile in HILIC.

The amino partition columns will do the same thing as the silica column, and the standard mobile phase is acetonitrile/water. On the other hand, you cannot use acetone as solvent for amino columns, unless you are planning to create a novel bonded phase.

All of these columns can use gradients, but then you can't use the refractometer for detection, and you are back to using acetonitrile. On the other hand, from the description of the problem, I do not see a need for a gradient: the contaminants that you mentioned will all give less retention in HILIC than ethanol. Then why would you need to run a gradient?

[adam]

Hmm. GC is starting to sound like the simplest solution.