Chromatography Forum

I would like to open up a discussion on this topic. What is the cause for the negative peaks in IP-HPLC? These negative peaks are not showed on the viod. Any comments about the cause or the ways to eliminate them. Thank you.

Is there a difference between your mobile phase and sample diluent?

AFAIK there is an equilibration state between mobile phase and stationary phase. When you inject something that differs from your mobile phase, (for example - stronger solvent) it will behave like any other analyte in column - will take part in partitioning between phases in purpose to reach equlibrium state, than your IP reagent concentration will partialy increase in mobile phase, and when it goes through detector, in case of lower absorbance vs. your mobile phase, you'll obtain negative peak.

The sample was injected in both water and mobile phase. No strong solvent was used for this. Any additional thought? Thank you.

Was the water the same ionic strength as the mobile phase?

Negative pekas in tru ion-pair chromatography are no unusual. The subject has been covered in the literature.

Roughly, if you inject analytes into an ion-pairing system, they will loose the counterion with wich they were injected, and acquire a counterion from the ion-pair reagent. Since the analytes now move some amount of the ion-pair reagent with them, this must leave a hole of ion-pair reagent somewhere. The hole of ion pair reagent is the negative peak, which moves with the same velocity as the peak from an injection of the ion-pair reagent.

Publications by several authors which argue with each other about minute details of the process.

Jan,

Provided that you've chosen a UV transparent ion pair reagent system (e.g. tetrabutylammonium phosphate or sodium hexanesulfonate) you shouldn't actually see any more negative peaks than you would in any other mode of chromatography. The general problem is that the purity of such reagents can often be rather poor and the impurities, if they are UV absorbing will cause the negative peaks you mention. I remember analyzing a batch of rather discolored tetrabutylammonium hydroxide from Aldrich for an ion impurities. To my surprise I found significant levels of not just the bromide from which it was made but also nitrate, sulfate and chloride. If eluents are prepared with such ion pair reagents seeing a lot of negative peaks is not surprising since both bromide and nitrate absorb in the UV. Of course, the other problem is that often buffers are constructed will UV absorbing ionic components and this also contributes to additional negative peaks. In my experience the best quality ion pair reagents are from Fluka and for tetrabutylammonium salts: SACHEM.