What is the optimal ratio of ion pairing reagent to use in an HPLC determination? For example, if I'm injecting 10 uL of a 5 mM solution of my analyte (looking for analyte impurities), what would the ion pairing concentration need to be in order to get good chromatography?

The standard ion pairing concentration is 5 mM. Anything from 3 to 10 is fine. What is your ion-pairing reagent?

I would like to ask an additional question. Anon from 26 Oct 2001, 2:44 pm said anything from 3 to 10 is fine. I am currently developing a method for glycolic acid using ion pairing chromatography and need to use 25 mM TBAHS. Is this too much? If so, what could be some problems from using such a high concentration? Any help would be greatly appreciated as I am new to ion-pair chromatography. Thank you.

There is no single "best" concentration of ion-pairing reagent.

To a good first approximation, as you add more TBA to the mobile phase, ionized acids will see a significant increase in retention, unionized compounds will see a slight decrease in retention, and ionized bases will see a signficant decrease in retention. The net effect is that you can "tune" your selectivity by adjusting the TBA concentration.

Some really neat work along those lines (including recommendations for IP reagent selection and nomograms for ajusting concentration) was done by Gyula Vigh at Texas A&M about a decade ago. It's summarized in the Snyder, Glajch and Kirkland "Practical HPLC Method Development" book. (Yes, we sell it:
http://www.lcresources.com/books.htm )

How high is "too high" depends on the organic solvent concentration. The Vigh nomograms suggest maximum TBA concentrations ranging from 10 mM (at 10% MeOH) to about 60 mM (at 50% MeOH). In any case, you want to stay low enough to avoid forming micelles!

-- Tom Jupille / LC Resources Inc.

Tom is right, you can use higher concentrations. It depends on the ion-pairing reagent that you use. The limiting factor is the formation of micelles. This occurs at lower concentrations with longer chain lengths. It also depends on the % organic. It may be good to look up the Vigh reference. Be careful though. If 60 mM is good in 50% MeOH, you may still get micelles at lower concentrations in a more aqueous mobile phase.