IOn Exclusion Columns

[josebenjamin]

Dear Friends,

Ion Exclusion columns are very limited in the range of conditions in which they can be used. Normally is only one mobile phase composition (little or no organic content), and a limited range of flowrates and temperatures.

Does anyone know of some procedure(s) to be used to regenerate their performance. As it happens, some lose resolution and sometimes they also increase backpressure. Any information on this will be highly appreciated.

Thanks,

josebenjamin

[Mark]

josebenjamin,
Your best bet is to contact either Alltech or Biorad and ask them. If back pressure is a problem, in my experience the column needs to be replaced. The polymer in the column is extremely fragile and easily crushed, leading to the high back pressure.

Regards,
Mark

[Chris Pohl]

josebenjamin,

The thing to keep in mind with columns of this sort is that they are a bit on the fragile side so cleaning protocols should be "gentle" but this does not they can't be effective. But, patience is needed since these columns have quite high loading capacity and it can take overnight or even a couple of days of cleaning to get all the garbage off a heavily fouled column. Thus, it's a good idea to target the cleaning with a bit of knowledge as to the nature of the contamination.

If the column pressure is high, it could be due to particulate based plugging of the inlet. In this case your best hope is to replace the inlet frit. If this doesn't reduce the column pressure, it's likely that the top of the column is contaminated with particulate. Just about your only hope in this case is to reverse the flow but make sure though column doesn't have any headspace before you do this, otherwise the column is ruined (although if the column is more or less dysfunctional at this point, there is little risk). Another possible cause of high pressure is contamination with "swelling agents". Such swelling agents include organic solvents and other organic molecules which might have a high solubility in the stationary phase and limited solubility in water. The cleaning protocol in this case is dependent upon the nature of the contaminant. If the contaminant is non-ionic or possibly a weakly acidic organic, the best strategy is to use an organic solvent rinse. 30% acetonitrile will generally not cause irreversible damage to such columns but it may take a day or two to clean the column and restore it back to original aqueous eluents. Keep in mind that such columns generally don't care for abrupt solvent changes. A gradient to and from solvent containing mobile phases is advisable. If the contaminant is possibly a cationic species, the mobile phase will also need to contain a substantial amount of acid in order to facilitate elution of the compound (although such compounds generally cause bed shrinkage and headspace and often such damage is unrecoverable). Transition metal contamination is best removed with a chelating agent which is acid soluble such as oxalic acid. Can you comment a bit more on possible sources of contamination?

[josebenjamin]

Dear Chris and Mark,

Thanks for your comments. Unfortunately there is no information about the nature of the contamination. Therefore I will need to try some "general" cleaning procedure.

Thanks very for the hints, I hope someone else can add to your comments.

Josebenjamin

[adam]

Just out of curiosity: what is the mechanism of separation with ion-exclusion columns. None of the textbooks I have mentions this technique.

[Steve]

Ion exclusion is when highly ionized samples are eluted first from the pores of the sulfonated poly(styrene-divinylbenzene) support then the lower ionic and nonionic compounds are eluted later. The separation mechanism is determined from the sample compound and sample conditions. Hydrogen bonding can be one of the seperation mechanism employed.

[Chris Pohl]

Adam,

First off, there are several good books on ion chromatography which explain the mechanism of this separation technique including: Ion Chromatography by Hamish Small (the inventor of ion chromatography), Ion Chromatography by Joachim Weiss (the third edition of this book was recently published in English) and Paul Haddad also has published a book on ion chromatography which covers this separation technique.

To add to what Steve mentioned below, I would describe the retention mode as non-ionic inclusion (generally separation techniques are named according to what is responsible for retention but in this case, curiously, the name Ion Exclusion describes the mechanism responsible for a lack of retention). That is, the stationary phase contains a high concentration of fixed anionic charges throughout the matrix which prevent permeation of anionic species. Molecules are retained to the extent that they are non-ionic in the mobile phase. Any molecule which enters the stationary phase will remain non-ionic while within the stationary phase since the pH in the stationary phase is approximately 0. Control of ionization through adjustment of the mobile phase pH controls retention. Lowering the pH decreases the ionization of a given solute, increasing the non-ionic fraction thus increasing the partition coefficient of the analyte.

[HW Mueller]

If you want to have a mathematical description check "Donnan equilibrium" in the internet or a physical chemistry text. An example of an unwanted ion exclusion: At pH ~7 anionic radioactive substances emerged in front of tm (to), while cationic species were strongly retained. The effect could be "lifted" by adding a salt to the mobile phase (0.1M NaCl, or phosphate buffer....).

[HW Mueller]

The weather is too hot here these days, I forgot the most interesting aspect of the example above: The column was a C-18.

[Victor]

Chris-thanks for a very nice description of the ion exclusion process.

I would be interested to know in addition why the pH in the stationary phase is approximately zero?

[Chris Pohl]

Victor,

The reason why the pH is approximately 0 stems from the concentration of sulfonic acid sites within the stationary phase. The resin used for such a column has a fixed site concentration of around 1.7 equivalents per liter. Considering the fact that the counterion for all of these fixed sites is hydronium, this means that the formal concentration of hydronium within the stationary phase is 1.7 molar. Even accounting for activity coefficients and the pKa of the sulfonic acid group, this should still put the pH within the stationary phase at around zero. Of course, such a low local pH is sufficient to fully protonate all but the strongest acids once they enter the stationary phase so one should consider analytes to be virtually 100% non-ionic within the stationary phase unless they are analytes which are very strongly acidic. This detail has been overlooked in most theoretical discussions of the technique.

[Jeane]

Dear, Chris Pohl

About stationary phase pH almost zero and it's overlook in most theoretical discussions... Do you have any scientific article or book chapter about it?

Thanks

Best Regards!

[Chris Pohl]

Jeane,

I'm afraid, I'm unaware of any references on this topic. As I said, this aspect has been unrecognized in theoretical discussions. A bit more discussion on actual experimental information can be found in the thread: "new thread on pH inside a sulfonated column"