Distinct RTs for free-base and salt forms of same compound

[pipettemonkey]

Greetings..

Given a lipophilic substituted imidazole (or amine) compound (pKa between 4-10) that exists in free-base form in one vial (dissolved in CH3CN) and in HBr salt form in a second vial (also CH3CN).


Is it possible to determine (by retention time) which vial contains the HBr salt and which contains the free-base using reverse-phase HPLC (any mobile phase)?

Thanks in advance.

[SIELC_Tech]

Check the following link it describes retention of amines and their salts by HPLC, (bromide ion is UV active at 210 nm so you don't need a conductivity detector). You can separate organic and inorganic ions in the same run using our Primesep technology:

http://allsep.com/Technology_Alternativ ... graphy.php
http://allsep.com/Technology_SeparationOfCompounds.php
http://allsep.com/makeChr.php?chr=Chr_032

[Chris Pohl]

Pipettemonkey,

In the first place, the simple way to differentiate between the two is to dip a pH test strip into each vial and find which one is most alkaline. Also, there's a good chance that the UV absorbance shifts somewhat with pH for such compounds so you could probably differentiate them with a UV spectrophotometer (you should at least be able to see whether or not the bromide is present that way). But, from a theoretical point of view, you should expect that peak shape distortion upon overloading would be significantly different when injecting the freebase form versus the bromide salt when using a reversed phase separation with an unbuffered eluent system. Of course, as Sielc_Tech suggests, you could also simply assay for the bromide ion by one of many analytical methods including ion pair or anion exchange chromatography. But, the retention time of the salt and the freebase will be the same in a reversed phase system so you can't separate them based on reversed phase chromatography, if that's what you were wondering about.

[pipettemonkey]

Pipettemonkey,

In the first place, the simple way to differentiate between the two is to dip a pH test strip into each vial and find which one is most alkaline.

But, the retention time of the salt and the freebase will be the same in a reversed phase system so you can't separate them based on reversed phase chromatography,

That is pretty much what I thought. To differentiate a base from its salt, Melting points, pH of solutions, solubility, etc, are methods that I put before HPLC.. Unfortunetly, the handle I use is quite representative of my level of clout.

If the salt would travel as an ion pair, then, perhaps, its rt would differ from the base (?) From this, I conjured up an aprotic solvent (THF, CH3CN, CCl4,) approach as a place to begin.

With this or any solvent system (unorthodox or otherwise), is such a task even theoretically possible?

[pipettemonkey]

I'm starting to become interested in Br- detection using reverse-phase chromatography.. UV detection is my only option.

What could I expect from a run on a reverse-phase HPLC system with (only) UV detection (210-215 nm), and CH3CN as the only solvent?

Thanks again.

[Bill Tindall]

you state that you want to "detect" bromide by reversed phase. It would be vastly simpler to add silver nitrate and look for a yellow precipitate.

If you want to determine bromide, reversed phase chromatography is a poor choice as there should be no retention of this species. Ion chromatography, titration, better choices.

If bromide is present as a simple salt it will migrate down the column independent of the base and it will probably come off in the void volumn.

[Kostas Petritis]

Pipettemonkey,

There is only one reverse phase column which can achive what you want (I excluded mixte mode columns).

You can separate your polar compound and your Br- by using a porous graphitic column (Hypercarb, Thermo-Hypersil). This column has the ability, under the appropriate conditions, to retain inorganic anions, Br- is one of them.

Here is a reference for you:

Elfakir et al. J. Chromatogr. A 829, (1998), 193-199.

Good luck!

Kostas

[pipettemonkey]

If bromide is present as a simple salt it will migrate down the column independent of the base and it will probably come off in the void volumn.

Would use of non-polar aprotic mobile phase (hexane, for instance) increase RT of Br- appreciably?

[Kostas Petritis]

Think about the Br- solubility in Hexane...

Furthermore, what are the polar groups in your reversed phase you are going to use for Br- retention? The only ones out there are the residual silanols...

[pipettemonkey]

The analysis of Bromide ion is somewhat of a digression from my initial posting interest. At this point, I would be pleased to see any Br- peak no matter how early it elutes following injection of amine-HBr salt- The other less likely alternate being ion pair migration of the amine salt.



Thanks again to everyone for the valuable insight.
This site is a real gold mine! Excellent commentary and informative archive.

[Chris Pohl]

Pipettemonkey,

As Bill mentioned in an earlier post, you will not observe retention of bromide in an analytically useful manner on a reverse phase column without addition of an ion pair reagent. The Hypercarb material mentioned earlier will produce some retention of ions such as bromide due to the fact that the material contains residual anion exchange capacity as a byproduct of the synthesis. So, the fact remains that you will need to add an ion pair reagent if you want retention of bromide on a true reversed phase column. Tetrabutylammonium ion will work well for this application (5mM should be adequate) with around 10% acetonitrile. Just make sure you choose a tetrabutylammonium salt which does not contain a UV absorbing counterion. From Fluka and SACHEM you can purchase tetrabutylammonium hydroxide of very good quality. If you neutralize this with methanesulfonic acid you will have a reagent suitable for UV detection.

Without any reagent additive, bromide will elute in the midst of the void volume disturbance and provide no basis for analytical determination. Making the mobile phase more nonpolar will not increase retention of bromide. Actually, if you wanted to increase retention of bromide you would need to add a high concentration of a non-UV absorbing high hydration energy salt in the hopes of "salting out" the bromide. Unfortunately, the only ion that I can think of that falls into this category is methanesulfonate and it would probably compete for surface adsorption, preventing any retention.

[HW Mueller]

pipettemonkey,
you can forget about an amine-HBr ion pair going through the column (formally, only if you have the amine in the mobile phase, which would be nonesense). The equilibria involved are so fast that either injecting the amine or the Amine-HBr will immeditly leave you with the same species as soon as they hit mobile phase (unless you overload drastically).

[Victor]

This is a very interesting discussion and I agree with everything that has been said-HWM has stated the practical difficulties very nicely. There is no mechanism to retain Br- so it will be eluted in the void volume of the column, and the base in whatever form it is injected will elute accompanied by the buffer anion-presuming that a buffer is used-which I think is essential to get meaningful results.

However, there is a point that pipettemonkey makes which is interesting. In an aprotic solvent I do not think that it is possible for BH+ and Br- to elute at separate times to preserve the charge neutrality of the system. In this case there is a theoretical possibility of a difference in retention time between the injection of a free base B and its salt BH+Br-. However, this is only theoretical because neither would show any retention in the pure organic solvents necessary. There would likely also be problems of solubility as others have mentioned.

[HW Mueller]

Victor,
if you assumed silica to be aprotic (and non-dissociative), maybe. But, if you had B, BH+Br- present, dissolved in an aprotic system, one should expect only one peak (if it chromatographed at all), as the equilibrium between B and BHBr should still be much faster than the chromatography.