Chromatography Forum

Hi All,
I will soon be working with an ion pair reagent (hexane sulfonic acid) on a C18 column, and since I have never worked with such reagents, only on conventional RP chromatography I an a bit bothered of how I should take care of my column in between use. How should I clean such a column after use before storage (even for prolonged periods), without shortening the column life??

Thanks for any suggestions
Omar

A good discussion on the subject can be found here:

http://www.sepsci.com/chromforum/viewtopic.php?t=6325

It is my experience that negative ion pair reagents can be relatively easily be removed from the column. In the case of long perfluorocarboxylic acid, I was able to remove them "completely" and had extended life times when using end-capped columns. By complete removal I mean that the column did not present any cation exchange properties anymore when compared with the same column before ion-pariing reagents are used.

For the regeneration conditions look at:

Petritis et al. J. Chromatogr. A 833 (1999) 147–155

I concur. Hexanesulfonate is quite easy to remove from columns. Just make sure your HPLC is well flushed too.

For overnight storage, I would keep the column in the mobile phase with the ion-pair reagent. The reason is that it always takes a while until the column is reequilibrated. How long is a while? This depends on the concentration of the reagent in the mobile phase...

I would agree with Uwe if you are working in isocratic mode, although I prefered to just decrease the flow rate instead of stopping it completely.

On the equilibration time issue, the equilibration time depends much more on the length of the hydrophobic chain (or the hydrophobicity in general of the non-polar part of the ion pairing reagent) than the mobile phase concentration...

It means that, the longer hydrophobic chain, the longer time I need to equilibrate the column? Or vice-versa?

Yes, the longer the hydrophobic chain, the longer the time you need to equilibrate the column (that is what you would expect with reverse phase). There are ways that you can calculate the exact equilibration time which varies depending on the reagent that you are using.

As I used perfluorocarboxylic acids in the past, I was able to calculate the equilibration time by monitoring the breakthrough curves with conductivity detector or low wavelength UV... I have some reference that I can give you if you want to see how they look like...

We could find certain recipe from Mr. Dolan in LCGC 24(9): 986 - 992, 2006 for removing ion-pairing reagents from the column. He recommended a 100 mL wash using a mixture of 200 mM phosphate buffer, pH 6, and methanol (50:50). But I do not know it will be work to which type of ion-pair reagent...