Ion pair reagent with gradient elutions

[bhaskarac]

Can any body explain me can we use the ion pair reagents in gradient elutions?
What may be the problems we face, if we use in gradient elutions?

[Kostas Petritis]

Yes you can,

Sometimes though, re-equilibration of a system is so long that makes the use of a gradient elution impractical. From a practical point of view, it is easier to use ion-pairing chromatography with shorter chain length ion pairing reagents or in the case of longer ion-pairing reagents smaller equilibration times are achieved with higher % of organic solvents.

At the end is the separation of your compounds that will dictate you which method will have to be used.

PS: Just for the sake of discussion, there is a study made in 1982 from some French group (L.E. Vera vila, M. Caude, R. Rosset, Analusis, 1982, 1, 36-42) where the authors said that: "(Ion pairing) chromatographic separataion with elution gradient will be reproducible only using isocapacitive mobile phases. (Isocapacitive mobile phases are defined as mobile phasaes with counter ion concentration corresponding to the same counter ion concentration in the stationary phase)". I didn't see any follow up articles on this subject from any other groups, maybe because the article was in French...

[Benjamin]

Dear Friend,

The use of gradient elution in ion-pair separations is perfectly feasible if you take some precautions. First, you must have exactly the same ion-pair reagent and buffer-salt concentrations in the mobile phases. It is also convenient to adjust pH (aqueous portion of each phase) to exactly the same value. Second, it is recommendable to run at least one or two gradient blanks before making any sample inyections. This allows the column to equilibrate. Third, the purity of the reagents becames critical under these conditions, and do not be surprised if you see significant differences in the quality of reagents. Fourth, most likely you will see some baseline "humps", these as long as do not interfere with your separation are not important. Fifth, try to use as high a wavelength as possible, all reagent purity gradients are more critical at low (<220nm) wavelengths. Sixth, try to dissolve your samples in the initial mobile phase if possible, even more, I recommend you use mobile phase that has been passed through the column for this.

Good Luck,

benjamin

[bert]

you use mobile phase that has been passed through the column for this.

Benjamin,

what do you mean by this?. The initial mobile phase or the final mixture after the gradient??

Regards Bert

[Kostas Petritis]

SIELC_Tech,

You really get annoyed when you see people asking about ion-pairing chromatography, don't you?

Although, I haven't used your columns (yet) they seem to be a good alternative to (some) ion-paring methods.

Now why someone would still use ion-pairing methods?

1) Because is a very well established chromatographic method for anionic/cationic compounds (a lot of methods already in the literature)
2) By manipulating the ion pairing reagent nature, it's concentration, the chromatographic support etc. you can achive different ion exchange capacities and achieve the desired separations and even selectivities you desire. For example, one can observe different selectivities by using perfluorocarboxylic acids or alkylsulfonates as ion pairing reagents, or by using different reversed phase material, or by using different alkyl chain lengths, providing the flexibility that someone wants for their separations. My personal experience is that you can achieve separations of very complex mixtures.
3) In most labs you can always find some anionic or cationic ion pairing reagents that you can readily use.
4) There are basic/acid volatile ion pairing reagents that can be used with ELSD or MS.
5) I browsed through your applications in your website and noticed that in most (if not all) the cases when the presence of acid is required (i.e. as an electronic competitor) you use TFA that although weak, it is still an ion-pairing reagent. At least, you have the same ion-suppression phenomena in MS as with any other ion-pairing reagent. Is it a reason that you use TFA for your application and not formic or acetic acid (i.e. better peak shapes or something else?).
6) I guess that there are facilities (i.e. universities) in some countries that an old horse is all they have to go to their work (although I have noticed your generous previous posts for free column for evaluation purposes and/or development of the method of interest in your facilities...).

Anyway, I guess I gave you "food" for more arguing, but I guess that this what this forum is about...

Note: The "old horse is all they have to go to their work" is a quotation I used from a previous post that was deleted from the administrator... I did not mean to offend anyone

[SIELC_Tech]

Kostas,

TFA is not a "must use" component in our mobile phases. The applications were created at the begining of our studies and now we are trying to rerun them. You can create same elution effect by using formic acid, ammonium formate, ammonium acetate if you have LC/MS application -just create a right ion-strength of the mobile phase. If you have UV application you can use sulfuric, phosphoric acid or phosphoric buffer. By changing the additive in the mobile phase you can change selectivity, so basically you have endless possibility for adjsuting your selectivity.
And I do realize that some methods (for example USP methods) are witten almost in "stones" and it is hard to change them.

[Kostas Petritis]

SIELC_Tech,

Did you delete your message before I post mine? My post now does not make much sense ... next time I will quote your whole message before I will reply

[SIELC_Tech]

Kostas,
The message was sensored/deleted by "administrator". But i got your point and hopfully you got mine.