I have to optimize an old method, using tetrabutylammonium as Ion-Pair reagent. I am wondering whether is there any difference in using tetrabutylammonium hydroxide instead of sulphate (or phosphate).
Does anybody knows which are the advantages and disadvantages (if any) of the different choices?

There can be rather dramatic retention differences between different salts of tetrabutylammonium ion. Even if one sets aside pH effects, this is true. This is primarily due to two factors. First, the concentration of tetrabutylammonium ion adsorbed on the surface of the column is dependent upon the counter anion. The highest adsorbed concentration will be observed with the least hydrated counteranion. And of course the local concentration of ion pair reagent on the surface of the stationary phase has a profound effect on retention. Using counter ions with valency > 1 will also increase the adsorbed concentration. The second factor is the eluent effect from the anions chosen independent of their effect on the amount of adsorbed tetrabutylammonium ion. For example, the retention of a divalent anion in the ion pair mode with tetrabutylammonium ion as the ion pair reagent will be much more dramatically influenced when the counter ion is phosphate than when it is hydroxide. Accordingly, if you make eluent contain only solvent and ion pair reagent (i.e. without added buffer) you'll generally observe the longest retention time with the hydroxide salt and the shortest retention time with the phosphate salt. Apparently the eluent effects of the anion greatly outweigh the local surface concentration differences for tetrabutylammonium ion in different salt forms. Of course, one can't use tetrabutylammonium hydroxide on silica reversed phase columns since this will cause serious damage. All that being said, if you are using a large excess of buffer relative to the concentration of the reagent you will probably see almost no effect since the buffer anion will dominate the process anyway.

Chris,

Thank you for that thoughtful and informative posting. I have used TBA very little, and I see in retrospect that I might have never succeeded with it had I not also been using a buffer in the solutions.

This leads me to my question for you: Have you a preferred source of TBA reagents -- i.e., clean ones? When I was working with TBA, I found that the worst aspect of those reagents was the amount of UV-absorbing crud in them.

I proposed to repurify the reagent, but that was rejected as inappropriate for a field lab. So I just had to use the best I could find. The method worked, but it might not have had I had to read a signal at lower wavelength.

I routinely clean TBAOH with a SPE cartridge. The baseline in a gradient run is almost flat (254 nm).

Of course, I agree that in many cases a buffer is required in ion pair work but for most of my work I am using tetrabutylammonium hydroxide as a reagent for separation of the salts of strong acids such as chloride and sulfate. Since these ions are fully ionized under the conditions of operation there is no benefit to adding a buffer (and a disadvantage: the buffer adds to the conductivity background). But of course in more typical applications where the analyte pKa is close to the operating pH of the eluent, a buffer is a must. As far as the best source of tetrabutylammonium hydroxide is concerned, the clear winner is: Sachem http://www.sacheminc.com/ They have several different grades but the best grade is their electronic grade as a one molar solution. It is pure enough that it often crystallizes to a solid-state at room temperature so you need to warm it up a bit to give back into a liquid state. I believe that Fluka may be selling this grade of reagent as well (product number 86832) but of course we buy from Sachem because as the actual manufacturer, their pricing is better.

Is thereany difference between using TBA as chloride or as phospate??
Wich is the pKa of the TBA??

TBA is a quaternary compound and as such has no pKa (it is a cation at all pH values). Regarding the question about if there's any difference between chloride and phosphate salts of TBA, that depends on your eluent system and your detector. The counterion will definitely affect your separation if no other anions are present (chloride will give longer retention times at pH 7 but at pH 4 phosphate will result in longer retention times). But if you have other electrolytes present at substantially higher concentrations, the counterion to the TBA will not result in significant changes in retention.

From a detection point of view, phosphate is preferred since it is UV transparent. If you are using the chloride salt and working at low wavelengths you will probably observe a negative peak in your chromatogram corresponding to the retention time of chloride. For this reason, it is generally preferable to use UV transparent counterions. Starting with TBA hydroxide is generally preferred since this gives you the greatest flexibility in choosing the counterion. For example, while chloride absorbs in the UV, methanesulfonate does not so you can neutralize your TBA hydroxide with methanesulfonic acid and obtain chromatography similar to that with a chloride counterion but still work at low wavelength UV.

Thank you very much for your answer. I wish to do another question: I am trying to separate some antibiotics in a single method, for all of them only with methanol or acetonitrile and buffer at pH 7 was necesary for resolution, but one (amoxicillin) needs ionic par. Can I use ionic par for all antibiotics??

Thank you
Norma Cavazos

I am trying to separate a very hydrophobic protein. Using TFA as ion pairing we have a very low recovery (7%) of the protein injected (may be the protein aggregate and become "sticky" into the column?. I want to replace TFA and try TEA or TBAP to overcome this problem. I need your help. Also what is the amount of TEA and TBAP usually added?
Thank you.