Chromatography Forum

Dear members of this forum,

I work with a sodium salt of hexansulfonic acid as a ionic pair agent only and I use an exclusive C18 column as in many opportunities it is recomended. As this kind of agent it is very difficult to clean from the column with a standard procedure of cleaning owing to the hard hidrophobic union with the C18 chains.
My question is about it is a bad idea suppose that the ion pair agent is joined to the column after a many analysis and prepare the mobile phase with the buffer and the organic component without ionic pair agent and get a good ionic pair chromatography analysis.
I would appreciate any advice,

Diego Delmonte

Hello Diego,

I would not suggest to do this. The reason is the following. Your are right with the statement, that it is sometimes really hard to get rid of ion-pair reagents from a C18 phase. Nevertheless you will get rid of ion-pair reagents by consequently rinsing the column with mobile phases that does not contain any ion-pair reagents. Hexanesulfonic acid is quite polar and thus will also be washed of the C18 phase by rinsing with a highly aqueous mobile phase. If you are just doing a few injections on such a column without ion-pair reagents in the mobile phase, you will still get a similar selectivity like having ion-pair reagents in the mobile phase, but it won't be very robust and reproduceable.

Dirk

How long does this memory effect for alkylsulfonates last? Hexanesulfonate dissolves readily in water; can one even observe two identical injections after the removal of the IP reagent?

I don't know if there is published anything about such a "memory effect. As mentioned before I won't suggest doing this. The longer the alkyl chain of the ion-pair reagent, the longer the reagent sticks to phase.

if there is no significant memory effect during the first few runs after the removal of IP reagent from the mobile phase, does this demonstrate that the reagent itself is easily removed from the column?..

Hello JA,
you are correct in your observation. If after only a few injections you start to loose reproducability then it is safe to say that the "memory effect" as you call it is gone. I can tell you that we have seen it in our laboratory.
fruther more you should not forget that we are performing absorption chromatography. The hexansulfonic acid is found in both the stationnary phase and also the mobile phase. removing it from either one of them will influence the separation, eventhou the major effect should be in the fact that it is washed away from the column bed.
basically, you have to still use the hexansulfonic acid salt in your mobile phase.
But this is not true for all compounds.I know of research done in the introduction of lyposomes inside the column in order to achieve new separations. Now those lyposomes are totally absorbed by the column and are there to stay.

Triethylamine is like that too. We made the mistake of using an old column for development and running of a method that included TEA in the mobile phase. When we tried the same method on a new column, we found that we had to 'condition' the column by recirculating TEA through it for a while before it would work. I'm still not sure how we validated that method in the first place, unless everyone started with columns that had seen some TEA...

Dear members of this forum,

Thanks for your oppinion which are very valuable for me. I am not very clear this very interesting subjet for me because I was reading the article "The cleaning and regeneration of Reversed-Phase HPLC Columns" from LCGC North America and in page 24 in the half of the medium column mentioned the effect of ion pairing reagents on the stationary phase. In this article mentioned two agents as octanesulfonic acid and tetraalkylammonium bromide. It is mentioned at certain concentration the columns become contaminated and cannot be regenerated to their original state. I would like to discuss this subject with everybody who are interested in this subject. I like the subject and I would like to learn more about that.
Thanks in advance for your help,

Diego Delmonte

This has been discussed not very long ago, some doubted the contention that ion pair reagents change a column reversibly. From my experience it appears that the permanent change "theory" could have come about by misinterpretations due to:
A). ions, including buffers are difficult to remove when you use pure water to wash the column, they disappear very fast when using sufficient concentrations of other ions in the water (the ion exchange people should be able to say more on this)
B). some chemical other than the ion pairing substances could have attacked the column, maybe the column was operated outside of the pH range. The ion pair reagent could then have been blamed falsely for permanent changes.

(I have no direct ecvidence for reversibility in this context, but I am at a loss to find chemical or other reasoning for a permanent change caused by ion pair reagents alone)

Tequila,

Regardless of what may have been published in the article to which you refer, the actual situation is that removal of an ion pair reagent from a reversed phase column is only problematic when the ion pair reagent is cationic (e.g. tetrabutylammonium ion). This is because cationic ion pair reagents can bind to the column through a combination of hydrophobic interaction and ion exchange interaction with surface silanols. I wouldn't say, even in this case it's impossible to get rid of all the ion pair reagent, just that it may prove to be a rather slow and tedious process. But for the case of anionic ion pair reagents, there is no analogous ion exchange binding process and therefore these reagents can generally be removed quite easily, especially small ones such as hexane sulfonate.