Any one can tell me how buffer and ion pair reagent function in Reverse Phase chromatography ? or any one can suggest good book on this topics ?

Buffers control degree of ionization of stationary phase as well as sample components, which in turn have a great effect on retention because protonated sample components can have vastly different retention times compared to unprotonated compounds. Buffers may also affect sample and column stability by affectig hydrolysis and other pH dependent reactions.

I was never confident that I knew how ion pair reagents really worked, so I'll defer to someone else for that answer.

Superficially, you can look at ion pairing as a superposition of ion-exchange on reversed-phase. The ion pairing reagent is adsorbed onto the surface. Retention of ionic compounds of opposite charge increases, retention of ionic compounds of equal charge decreases. The interpretation becomes a bit more complex, if one realizes that charge balance must be maintained. This forces at least partial comigration of the ion-pair reagent with the analyte ions. This has been used as a visualization technique for non-UV-absorbing ions in the presence of a UV-absorbing ion pair reagent.
In my opinion, one of the best explanations can be found in the following publication: B. A. Bidlingmeyer et al., J. Chromatogr 186 (1979), 419-434

One doesn´t see much on retention of the ion pair which will be present in the mobile phase to a greater or lesser extend. Is that mechanism disproven?

Hans: are you talking about the comigration of the reagent with the analyte ion? This is part of the subject of the publication that I mentioned.

Uwe,
yes, have to check your ref.
On the comigration of ions to detect an analyte ion: Actually there should always be other ions present which can keep up the charge balance. It seems if there is some comigration there must be an equilibrium between associated and dissociated species. Then, if there are associated species (reagent and analyte ion) present in solution they have to influence the chromatography. It appears that your example (identification of analyte with reagent counterion) is proof for a (partial?)comigration mechanism of retention in such a case?

Hans:

As Uwe mentioned above, the article from the Bidlingmeyer cited above discusses and largely disproves the "ion pair" model. Bidlingmeyer cites several old references demonstrating conditions required to achieve ion pairs in solution and in essence these references prove that in the bulk solution of the mobile phase the dielectric constant is not low enough to support formation of ion pairs. This work showed that even pure acetonitrile does not have a low enough dielectric constant to support formation of ion pairs in solution and this is certainly not possible in the presence of water. What you may be remembering from the literature is that, when one uses a "surfactant" ion pair reagent there is often a maximum in the retention vs. ion pair reagent concentration plot. This maximum is associated with formation of micelles in the mobile phase. In this case, there is competitive association with absorbed surfactant on the surface of the stationary phase and surfactant micelles in the mobile phase. Once the concentration in the mobile phase exceeds a critical concentration such that the stationary phase is "saturated", further increases in concentration only increase the micelle population in the mobile phase and thus association with the mobile phase population of micelles reduces the association with the stationary phase. But in the more common case where the ion pair reagent is not a surfactant this phenomenon is not observed. For example, tetrabutylammonium hydroxide does not really qualify since it is symmetrical and exhibits minimal surface tension lowering properties and certainly so-called ion pair reagents such as perchlorate and trifluoroacetic acid have no surface-active properties. None of these species exhibit a pronounced maximum in retention due to the micelle effect mentioned above.

If the organic component of binary mobile phases adsorbs to the stationary phase and forms a nearly pure organic layer at the surface of the stationary phase, as I recall from some of Pemberton's work a couple of years ago. Then, can't many of the ion pair reagents be viewed as phase transfer catalysts?

Chris gave a nice review. No need to add anything.
Hans: comigration is caused by the requirement for charge balance, but it does not mean that there is an ion-pair.

A cursory reading of the Bidlingmeyer article has not helped, I have to dig into it somewhat deeper. I am troubled with his proof against ion pairs in solution via conductance measurements. That certainly wouldn´t "see" very small equilibrium amounts of ion pairs. Minute amounts of ion pair in equilibrium could strongly influence the chromatography, though, theoretically. Also his statement "The net result is that a pair of ions (not necessarily an ion pair) has been adsorbed....." is less than overwhelming.

Uwe,
I need to take a pencil and paper and play through some ion balancing, or is there a very simple "picture"?

The article has a very intriguing discourse on peaks ahead of the dead time. If I recall correctly, we have seen this, not only with proteins, but with small neutral molecules (think it was ouabain) which had reacted on the column, maybe to ionic species??. Certainly will look at that.

Hans,

Perhaps the article Bidlingmeyer by itself isn't totally convincing but if you dig into the cited references you'll see some very convincing data on the matter. Using conductivity detection, you can detect with good sensitivity even small traces of ion pair formation.

But, as Tom suggests above, while the scientific data does not support the presence of ion pairs in bulk solution, it is not unreasonable to think of the electrical double layer model and the ion pair model to be different ways of describing the same system provided the scope of the discussion is limited to the state of ions within a few angstroms of the liquid-solid interface. It's true that surface science has a well-established model system to describe the nature of the interface and it's associated net charge. But it's also true that the dielectric constant at the interface is most likely sufficient to support the presence of true ion pairs, at least under some conditions. So, while the typical model of the interface has absorbed ions on the surface and co-ions immediately above the surface, it's definitely conceivable that there are also ion pairs on the surface as well.

One bit of evidence that would tend to support a partition equilibrium for the ion pair rather than an "ion exchange" equilibrium for just co-ion can be seen by doing breakthrough curves with tetrabutylammonium salts. In a simple ion exchange model, one would expect chromatographic retention of one co-ion to be retained longer due to a more compact electrical double layer. But the fact is that if you compare, for example, tetrabutylammonium nitrate and tetrabutylammonium bromide breakthrough curves, you will see that a larger number of equivalents of tetrabutylammonium nitrate is absorbed under identical conditions. In fact, the amount absorbed correlates precisely to the retention order for a series of monovalent anions.

Of course, the electrical double layer model also supports this observation since a less hydrated anion such as nitrate will permit a more condensed electrical double layer, which in turn supports a higher concentration of adsorbed species upon the surface. It's just that an "ion exchange" model for the system is a bit misleading since both retention and capacity are increasing at the same time in this system, which is quite distinct from a real ion exchange system (there are a number of other distinct differences between ion exchange and ion pair including selectivity for polyvalent species, for example).

So, I guess what I'm saying is that, the ion pair model is a lot easier to conceptualize than the electrical double layer model and to first approximation (as long as you are limiting your thinking to the area a few angstroms above the surface and the surface itself, rather than the bulk solution where, typically, no ion pairs can exist) the ion pair model still works equally well in allowing the practitioner to predict from physical properties the effects of experimental variables. As such, I see no reason why one can't continue to conceptualize the process as involving ion pairs.

Thanks Chris,
so this is not a "closed case" for partition. If there is unequivocal evidence that the solution has no ion pairs, than it is clear that the conventional ion pair explanation is out. However, can one be sure that the common ion pair reagents do not form ion pairs with at least some small analytes? (The evidence for ion pairs in solution of proteins seems overwhelming).

I just like to caution against the widespread tendency to overgeneralyze.

Can anyone suggest a good reference as a starting point for anion exchange chromatography?

el2

Can you be a bit more specific? It's a bit hard to generalize on such a broad topic. What compounds are you interested in separating? What detectors do you have available? Is your system stainless-steel or PEEK?

what is the role of buffer & ion rairing in ion exchange chromatography?

The answer is well covered in the earlier part of this thread. But, to grossly oversimplify:

- the function of the buffer (in the strict sense of the word) is to maintain a constant pH in the mobile phase, no matter what the sample.

- the function of the salt (often also called the "buffer") is to provide a source of ions which will compete with the analyte ions for access to the ion-exhange groups.

- "ion pairing" is, strictly speaking, an alternative technique to ion exchange.

For a more detailed discussion, see chapter 7 of "Practical HPLC Method Development" by Snyder and Kirkland.