Ion Pairing

[EyD]

I'm fairly new to ion pair chromatography (Decane Sulfonate of Na). I don't have a clear idea of the best conditions of washing and storing the column for short and long periods while the column remains unused.

[Rob Burgess]

We find the biggest problem to be washing IP reagents from the HPLC system itself. We routinely wash the entire HPLC system with IPA / water (10/90 v/v). That should be OK for the column also.

Is there any real side affects for leaving the IP reagents actually in the column? I read frequently that once you have used IP reagents with a column then column should be dedicated for IP chromatography. Does anyone confer with me on this?

[Consumer Products Guy]

A problem with leaving ion-pair solvents in the column is that the solvent will dry up and leave the ion-pair reagent crystals stuck in the column. I'd rinse with similar mobile phase mix that has the ion-pair reagent omitted. And sililar would happen in your HPLC plumbing, pump, injector if not rinsed. So either rinse or keep the flow pumping constantly at a reduced rate.

[ravenwork]

Good topic,

We have an ion-pair analysis that we do infrequently. Consequently, I am not comfortable storing the column with any of the ion-pair mobile phase. The mobile phase we use contains Na-hexanesulfonate, phosphoric acid, and diethylamine. It will grow mold very easily. I try to keep the mobile phase between runs since the Na-hexanesulfonate is expensive, but I had to dump some today because it had been over a month since the last run, and there was mold floating on the surface.

I really don't want anything growing in the column or the system. Thus, I flush the system and column with water and then store the column with 50:50, water:ACN. This requires a bit of a long re-equilibration for the column, and I always have to watch the first few injections. If the peak is split I know it probably hasn't re-equilibrated with the ion-pair reagent.

ravenwork

[EyD]

I mostly agree with you Ravenwork but I still have some doubts.
I work with this mobile phase : Acetonitrile 13%: Buffer(phosphate pH 6,6) 87% and the IP reagent (Na decanosulfonate) in low concentration, 2 mM.
I've been washing the column with methanol/water (70:30) to store it from day to day, and for longer periods of time.
Last time when I wanted to perform the analysis, I got only split peaks, even though I let the column equilibrating for many hours.
I don't know what happened, I'll give it a try to your suggestion of washing and storing the column with water: acetonitrile.
But how can I realize that the split peaks are due to a bacterial contamination or that the column is damaged or something else.

[ravenwork]

Well, there certainly are other possibilities for split peaks.

If I see splitting due to an unequilibrated column, it usually only takes another 20 columns volumes beyond my usual to correct the problem. If it were to go longer, I would suspect other things.

An easy thing to try is reversing the column. If this fixes it you probably have a fouled frit. I have gotten a fouled frit prior to improving my column and system cleaning techniques. But, I run with 100% aqueous which exposes me to microbial action. I don't think a 13% ACN mobile phase would have microbe problems.

Another source is samples that aren't properly treated with the ion-pair reagent. You might try adding a small amount of the ion-pair solid to a portion of your sample and see if it corrects the splitting.

The multiple equilibria involved in ion-pair chromatography can be a complex monster. It's a wonderful thing when it works, and a mind-grinder when it doesn't.

ravenwork

[Uwe Neue]

With 13% acetonitrile, I am not terribly concerned about bugs. Since you need to run the column the next day, I would keep it in mobile phase, since it takes a long time to reequilibrate the column with the ion-pair reagent.
If you plug the column properly, I would also not be concerned about the column drying out over longer periods of time. I would leave it in the mobile phase even for longer storage.

[syx]

Is there any real side affects for leaving the IP reagents actually in the column? I read frequently that once you have used IP reagents with a column then column should be dedicated for IP chromatography. Does anyone confer with me on this?

I have washed my column that used with IP reagent before. When I use the column to other method that not using IP, the chromatogram was different than in ‘normal’ condition, especially for ionic substances. I think the IP reagent could not be removed completely from the column.

[Chris Pohl]

syx,

I'm sure this has been discussed before but ease of removal of an ion pair reagent from a reversed phase column is highly dependent upon the charge of the ion pair reagent. Negatively charged ion pair reagents can generally be quickly and completely removed with simple washing procedures using mobile phases with a relatively high organic solvent content (e.g. 80% acetonitrile). The problem is with removal of positively charged ion pair reagents such as tetrabutylammonium ion. The issue is that these reagents tend to bind to residual silanols in silica based reversed phase columns. Both negatively and positively charged ion pair reagents can easily be removed from polymeric reversed phase columns.

[sneha]

i prefer using water :acn 80:20
or Acn :water 80:20 depending on ion pair reagent used.
it works best for cleansing but takes 1hr next day to achieve equlibrianext day.