Chromatography Forum

I have a compound that forms an enolate and therefore require the use of Ion-Pairing HPLC. Most literatures suggested the use of tetrabutylammonium phosphate in methanol and PIC Reagent A (manufactured by Waters) in water.

However, both TBA phosphate and PIC Reagent A are expensive to get.

I'm wondering if anyone out could help me out with a few ion-pairing basics?

1) What is the reason behind using TBA? My supervisor suggested the use of ordinary cationic salts (eg ammonium) to produce the charge. I personally don't think it's going to work, there must be a reason behind why people are sticking to the use of TBA and not cheaper chemicals. Does anyone know why?

2) What is the main difference between TBA phosphate and TBA sulphate? The latter is cheaper for some reason.

3) Does anyone know what is contained in the PIC Reagent A? As far as I know, it is basically TBA phosphate buffered at pH7.5. Is is possible to make our own rather than paying over $100 for 25 ml?


Would appreciate with any contribution!

Thanks in advance.

Judy

Generally, ion-pairing reagents are surfactants. This is an oversimiplification, but you can think of them as having a charged end, which interacts with the analyte, and a hydrophobic end which interacts with the stationary phase.

Ammonium ion can act as a weak ion-pairing reagent, but it certainly would not be a direct substitute for TBA.

The ionic form of the ion-pair reagent is, to a first approximation, irrelevant. Once in solution, it will be dissociated. Generally, you would want the TBA to have the same counterion as the buffer you will be using, hence the preference for the phosphate salt over the sulfate. The alternative is to use the hydroxide, which is then neutralized when you make your buffer.

There is nothing "magic" about the Waters PIC reagents. You're being charged for the consistency and convenience.

Dear Tom,

Thanks for your reply. That was very helpful, I got to read a bit more about ion-pairing chromatography based on what you told me. I guess I neglected the importance of the hydrophobic tail of the IC reagents which turns out to be essential in the retention.

By any chance, does anyone know what is the concentration of TBA phosphate in the Waters PIC reagents?

Most literatures seem to make up the TBA in methanol from scratch, but use the Waters PIC Reagent in water. Is there a reason why they can't make up the TBA in water themselves?

Thanks again.

Sorry, I don't know the concentration, but it's got to be in a spec sheet somewhere.

As to making up TBA phosphate in water, I would imagine that having carbon, nitrogen, phosphorus, and water, it would grow bugs fairly quickly.