Chromatography Forum

Dear colleagues,

I have one practical question for you and I'll really appreciate your input or shared experience.

Is it feasible/recommended to change between alkylsulfonate ion-pair (IP) reagent and tetraalkyl ammonium IP reagent on the same column?

What's my real situation now: today, I used heptane sulfonate in my mobile phase for impurity determination in pharmaceutical product. For that purpose I used C18 Discovery 250mmx4,6mm i.d. 5um column, which was already "dedicated" for ion-pair chromatography methods that use alkylsulfonates as IP reagents. After this analysis I plan to "regenerate" the column with phosphate buffer pH2.5 : methanol = 50:50.
I know that the use of ion-pair reagents can alter the initial column separation characteristics (that's why we dedicate columns). Tomorrow I'll need to use same (similar) C18 column to run another impurity method that uses tetrabutyl ammonium hydroxide as IP reagent in the mobile phase.

The question is: Should I use the same "dedicated" and "regenerated" Discovery column or to use another equivalent? We would like to avoid running this analysis on new (IP reagent "not contaminated") columns, as we rarely have the need and oppurtunity for running ion-pair chromatography methods. Is there any special precautions that I should be aware of if I continue to use the same column?

Your input is highly appreciated.

Regards

You are trying to switch between two ion pairs of totally different polarities ----

Sulfonate - a negative ion pair
tetrabutyl ammonium - a positive ion pair

Specially, if you are looking to quantitate impurities - my answer is a definite No-No!

Based on our experience in the lab with regenerating ion-pair columns - is extremely tedious and is a waste of valuable time.

Even if it seems expensive to buy a column or use a newer one - I would rather take this choice than trying to regenerate ion-pair columns.

Just a hunch: I would guess (probably wrong) that one could "regenerate" a column quite rapidly to use with ion pair reagent of the opposite charge.
zokitano, why don´t you just run your alkylammonium mobile phase on your sulfonate column and tell us what happened.

I use a single Luna C18(2) column for much of my quantitative work.

The ion-pair mobile phases range from tetraalkylammonium reagents to alkyl sulphonates, as well as a wide range of reverse phase buffers.

I have no problem washing the column to remove ion-pair reagents. Yes, it can take about 45 minutes instead of 15 mins, but I see no loss of performance. I've used both TBAH and octyl sulphonates ion-pair reagents on the same column and same day.

There may be good reasons to have multiple dedicated columns if you are routinely using them, and have a generous budget, but I think your system will work fine. You just need to allow for the longer equilibrium times.

My only proviso for you example would be the flushing. I prefer to wash columns containing ion-pair reagents with both high water and pure acetonitrile mobile phases after the buffers have been removed.

Some people have suggested using 1:1 MeOH: pH=4 100mM potassium phosphate buffers for flushing terabutylammonium compounds ( PO4 reduces column interaction ) , but I tend to just use copious amounts of acetonitrile and water. The sulphonates usually require a high organic water phase ( eg 80:20 MeOH:water ), but no buffer.

Note that, for some chiral columns ( eg Lux ) , some ion pair reagents certainly can affect the long-term performance, and it's sensible to dedicate those chiral columns - especially if resolution is important, otherwise you'll see a drop in performance after the first few times, then column performance will stabilise..

Please keep having fun,

Bruce Hamilton

I suppose there's a case for saying that if your two ion pair reagents have opposite charge, then if you don't remove every last trace of one, it is unlikely to interact with the analytes for which you use the other anyway, so perhaps it's less serious than trying to use two ion pair reagents of like charge but different hydrophobicity on a single column. But I might be writing rubbish, especially if you have analytes with both basic and acidic groups.

On the subject of keeping a column for each ion pair reagent: if you work in a lab where there is no pump spare for regeneration, you have to consider machine time regenerating. If you change ion pair reagent frequently, and your instruments are working on samples most of the time, it may be economically sensible to keep a different column for each ion pair reagent: the instrument time lost in regenerating may, over the lifetime of the column, cost more than the column anyway.

Thanks to all for your insights and recommendations.

I have used the same C18 (Discovery) column for both heptane sulfonate and tetrabutyl ammonium ion-pair chromatography analyses. It passed very well indeed. The time needed for my HPLC column to stabilize with tetrabutyl ammonium mobile phase, after its regeneration (after using the heptane sulfonate) was about 2 hours at 1,5 ml/min. The baseline was good and most important, the chromatography was excellent

The system suitability requirements for both ion-pair impurity methods were also met, and after the last analysis today I have "regenerated" the column again following the procedure suggested by Bruce.

Just to made one final remark: I had to switch to the positive counter-ion IP reagent in my mobile phase, only a day after the column was used with the negative counter-ion IP reagent.

Best regards