Chromatography Forum

Hello! I'm brand new to HPLC and was very excited to test out a column today. The run went pretty well and we were going to do another one, but when we ran the buffers through the column to clean it, we started getting weirdly high absorbances (1.5-2 range) at 260nm. As my buffer A, I'm using 0.1M TEAA and as buffer B, I'm using 0.1M TEAA + 25% acetonitrile. It's not the column, because running the buffers straight through the UV detector also gives these high absorbances. I also don't think it's something in the line because if I take a sample directly from either buffer bottle and spec it, they both absorb high. Basically, I'm wondering if TEAA is light or temperature sensitive, thus explaining why everything was ok at first but then went to hell. Anyone who uses this buffer system (mostly used for oligo purification) seen this before?

The purity of the triethylamine is critical. You may need to evaluate several sources before finding one that is good enough for this assay. I had good luck with Atofina, but there may be others too.

Also, the 25% acetonitrile should be specified to +/- 0.1% if you want repeatable retention times. If you are making your own mobile phases, do it gravimetrically.

I am confused by the nomenclature of TEAA. It seems to be interchanged between the quaternary cation tetraethylammonium and trisubstituted triethylammonium (acetate).

Looking somewhere like Sigma gives the impression the triethyl- derivative cannot be purchased in solid form, while the quat salt can. I also misunderstand how triethylammonium acetate can be used as a buffer at pH 7-7.5 (seen in previous Chrom Forum threads) when literature lists pKa for Et3N+H as 10.75.

Some other things playing on my mind over these two compounds are as follows;
- Does triethylammonium acetate have any advantages over solutions of triethylamine as a buffer? Is it any more stable? (I've got anecdotal evidence of diethyl- and triethylamine going brown on storage which I think gave high background in UV).
- Are tetraethylammonium salts commonly used as ion pair reagents or do the tetrabutyl equivalents predominate? Is the choice of one over the other useful in a method development sense?
- Are the triethyl- or tetraethylammonium salts sufficiently volatile for compatibility with evaporative-type detectors (ELSD, ESI-MS)?

Thanks

JA

You are correct, triethylammonium acetate cannot be purchased in solid form and is sold in solution, usually at concentrations of 1 or 2 mol/L.
It is not used as a buffer but as an ion-pairing reagent. Main application is separation of nucleic acids (oligonucleotides, RNA and dsDNA). It is volatile so it is MS compatible.
pH is of the mobile phase is somewhat critical, at pH>7 baseline absorption increases due to more free triethylamine which has significant UV absorbance.

As Mark pointed out, the quality of the triethylamine is important and you might have to shop around to get the quality you need. Double or triple distilled triethylamine is best.

cheers

The tetra-alkyl ammonium salts are not volatile enough for ELSD. The choice of Et4N+ versus Bu4N+ depends on the anion you are trying to retain.

I've got to ask Mark, based on your open-ended reply, if you might elaborate on the choice of tetraethyl- and tetrabutylammonium salts for some example solutes? One gets the impression you may infer that one or other quat may be unsuitable for a given analyte.

As I haven't played with these reagents, can anything be said of the likely changes in retention for a given analyte, let's say, some typical amino-functioned hydrocarbon. Is it as simple as longer chain, more retention? That's the experience we've garnered through use of alkylsulfonate homologues - although I'm yet to understand why. This is probably due to my uncertainty over the numerous mechanisms proposed - ion pairs in the mobile phase, dynamic ion-exchange and variations on electrical double layer theory