Chromatography Forum

Dear all,

I'd like to ask, what is the most suitable column cleaning procedure to remove ion-pairing agent from RP column since the agent might be retained strongly in the column? Are there any differences in the procedure for removing positive (such as alkyl ammonium salt) and negative (such as alkyl sulphonate salt) ions?

Any suggestions would be highly appreciated

See if this article helps:

http://www.lcgceurope.com/lcgceurope/da ... rticle.pdf

Ionic liquids have been a hell to remove from both silica based RP and "HILIC" columns. From this I would judge that sulfonates would come out best under basic conditions and ammonium species at acidic conditions. If one doesn´t want to have even "simple" positive ions, like Na+, stuck on the column one may use acidic conditions (enough acid to obliterate the silanolates) and a high ionic strength to get rid of both unwanted positive and negative species.

See if this article helps:

http://www.lcgceurope.com/lcgceurope/da ... rticle.pdf

I've read the article and found that,
"Ion-pairing reagents such as octanesulphonic acid (used for cations) and tetraalkylammonium bromide (used for anions) strongly sorb on the surfaces of bonded-silica columns at certain concentrations of organic modifier. The columns become contaminated and cannot be regenerated to their original state, and the story goes that any column used for ion-pairing work should be dedicated to that technique and never used again for regular reversed-phase chromatography."

Actually I've realized the fact that the column can't be used for regular RP chromatography.

However I've found another article that stated, "For removing ion-pairing reagents, wash the column with 100 mL of 200 mM phosphate buffer pH 6 mixed 50:50 with methanol." There are no differences for removing either anions or cations.

I just wonder if there are any other column cleaning techniques for removing such ion-pairing agents in case we have to reuse the column for regular RP chromatography.

From this I would judge that sulfonates would come out best under basic conditions and ammonium species at acidic conditions.

Do you know how I should run the column cleaning procedure (more detail explanation if you don't mind)?

TNA:

The next sentence in that article referred to an article (B.A. Bidlingmeyer, J. Chromatogr. Sci. 38, 226 (2000).) wherein the author suggested the following for cleaning columns used with ion-pairing agents:

"Bidlingmeyer9 disagrees with this generality [columns become contaminated and cannot be regenerated to their original state, and the story goes that any column used for ion-pairing work should be dedicated to that technique and never used again for regular reversed-phase chromatography] and feels that the aggressive pH values used for the ion-pairing coupling can actually change the nature of some columns by either hydrolysis of the bonded phase or endcapping silane under acidic conditions (pH 1–3) or by silica dissolution at higher pH values (pH 7–8). To remove sulphonic acid ion-pairing reagents, he recommends first washing the column (minimum of 20 column volumes) with the same mobile phase without the ion-pairing reagent and then washing with mobile phase without the buffer (methanol might be a better organic solvent than acetonitrile in this wash step; for very long–chain ion-pairing reagents, use tetrahydrofuran). Apparently, sulphonic acid ion-pairing reagents and amine ion-pairing reagents exhibit different behaviours. Bidlingmeyer and co-workers10 demonstrated that when using a C18 column with mobile-phase concentrations greater than 70% methanol, SDS, which is a long-chain anion-pairing agent, is not
adsorbed onto the stationary phase. This finding agrees with the Separations Group work.7"

From the above quote, I assume that if the use of ion-pairing agents has altered the stationary phase somewhat, your selectivity may be different than a new column of the same type that hasn't been used for ion-pairing with 'aggressive pH values' (I believe this is similar what Uwe Neue is referring to as 'column conditioning' in his HPLC Troubleshooting Guide - http://www.waters.com/webassets/cms/lib ... a20769.pdf).

Yes, it is clear that Majors is not one of those who think that detergents can not be removed.
TNA, you just prepare a high concentration buffer, or maybe add some salt to a low concentration buffer, making sure that there are no ions which tend to cause pecipitation (for instance, LiDS is more soluble in H2O than NaDS, but that should not be of much impact on concentrations encountered in washing).

I think the point to "dedicating" columns is not that you *cannot* remove all traces of the IP reagent, but that hard to be sure that all have been removed. By the time you fuss around figuring out a specific cleaning procedure, it can be more cost-effective to simply buy a new column.

Columns are cheap, time is valuable.

Thanks for all the replies. I think it's better if we "dedicate" the column for ion-pairing usage only in order to ensure its separation (comparing to other RP columns which have never been used for ion-pairing agent).

We have adapted this "principal" in our daily works. Therefore we have several groups of column which each is dedicated for certain ion-pairing agent. What we've experienced in the last several days is we had to use a regular RP column for our analysis because the "ion-pairing-dedicated" column showed bad performance. Hence I wanted to know how to remove ion-pairing agent from the column.

The other issue in all of this is cost-effectiveness. Take a look at how much your time costs versus the cost of a new column. "Your mileage may vary", but in many cases it's cheaper to simply get a new column rather than fuss with cleaning an old one.

"Columns are cheap; your time is valuable."

Thanks Tom ... I've got the point