It is the first time that I have to work with an ion-pair HPLC method and I do not know too much about it.
I have already looked to the LCGC articles subject-summary and choosen some interesting article references, I will try to get them, but I would be very greatful if anybody could explain "in some words" how does it works, when is it used, what kind of ion pair reagents do exist, etc..., where could I read about its theory and practice (I do not have Snyder & Kirkland).

Thanks in advance!

I'm sure others will give better explanantions, but here's my attempt at an explanation: Ion-pair chromatography's function is to separate charged species on a reversed phase support through the use of an ion-pairing (IP) agent. One dissolves a quantity of IP agent into the mobile phase and allows the system to equilibrate. This equilibration usually takes much longer (up to 100 column volumes) than a normal equilibration because the IP agent actually remains on the column during the course of the separation. The hydrophobic portion of the IP agent embeds itself in the column with the hydrophilic (charged) portion exposed and the separation occurs much like an ion exchange separation. Anionic analytes require cationic IP agents such as aliphatic tertiary amines and cationic analytes require anionic IP agents such as aliphatic sulfonates. There are a few general rules you need to follow when doing this kind of work. First, the pH of your mobile phase must be adjusted such that your analyte is charged. Second, the amount of IP agent you use is dependent on two factors. The first is the % organic in your mobile phase. The higher the organic content, the more IP agent you need. The second is the size of the aliphatic group on your IP agent. The more carbons there are on your IP agent, the less of it you'll need. Also, if at all possible avoid gradients or at least make sure they're shallow. If they're too steep and you don't have enough IP agent present, it'll wash off the column, your chromatography will be frustratingly irreproducible, and your equilibration times will be *very* long. If at all possible, dedicate the column you're using to IP mobile phases. As a matter of fact, if you need to use both anionic and cationic IP agents (in different methods of course) dedicate a column for each type of application. Upon completion of your run, you'll need to rinse your column carefully. I would recommend a first rinse of 50/50 MeOH/Water (NO buffer), then a good long rinse of neat MeOH or ACN. I like MeOH better because the anionic IP agents I use dissolve better in it than in ACN. My only other suggestion is to get a copy of Snyder and Kirkland and read the explanation there thoroughly. It's an extremely worthwile book for any practicing chromatographer in my very humble opinion. I can copy and fax a couple of pages out of mine if you like. Contact me by e-mail if you have any further questions. Best of luck and I hope this helps!

Chris

Oh yeah, another thing I forgot: There are now many columns on the market with imbedded polar groups or with silica (or polymeric) supports that are stable at high pH's, often obviating the need for IP agents. Many of these columns also run well with 100% aqueous mobile phases, so you may wish to find one of these columns before going into ion pairing. I would recommend speaking to your favorite supplier of columns and give them the particulars of your application. Very often they can be extremely helpful.

Chris

Chris, thank you for the explanation! I will have to "digest" it and read some more about the subject.

The compound that we have to develop a method for is a hydrochloride monohydrate. Even without knowing about ion pair chromatography I was thinking to try to ionize it with a basic mobile phase and see what happens. I think you suggested the same. Is it possible with a hydrochloride compound also?

Where could I find those old LCGC articles that I chooed from the subject index? We only have received the magazine for 1992 and there are articles from 1984-85.

I hope I will get soon Snyder & Kirkland - we are going to purchase it, so thanks for the fax idea, too.

Hmmmm...Others will hopefully correct me if I am in error, but I would be inclined to wither use a low pH and an aliphatic sulfonate (like Na+hexanesulfonate) or, depending on the pK, go to a high pH with a column resistant to high pH mobile phases. Definitely contact a column manufacturer tech support. I have gotten many good ideas and applications from them. I've had particularly good luck with the folks from Phenomenex, they're very helpful and I think they're a decent outfit. I've also had good luck with Waters, Metachem (Ansys?), and Supelco tech support. Re: the LC-GC mags, I haven't got any from the 80's - before my time! The fax offer stands, drop a line via e-mail if you have need.

Please let us know how things go!

Chris