I have to analyse by HPLC two molecules : gramine and hordenine which both present a tertiary amine. Their pKa is close to 10.
I found a protocol which describes the separation of both molecules using a phenyl column (so an apolar stationary phase) with as eluent a mixture of acetonitrile and phosphate buffer pH 7.2 supplemented with triethylamine 0.1%.
At pH 7.2, the molecules are cationic since the acidic form of the tertiary amine is the predominant one. Consequently, these cations will be poorly retained on a apolar stationary phase.
In my opinion, i would add a pairing agent (hexane sulphonic acid for instance, which is anionic at pH 7) to form a zwitterion with the cations, thus increasing their retention on the column.
So what is the usefulness of triethylamine (which is also a cation at pH 7) in the eluent ?
I think that triethylammonium cations block underivatized silanol groups which would strongly interact with your molecules of interest. For fresh endcapped column material you might not need TEA but it probably does not hurt. Adjust or check the pH after addition of TEA.
In addition to the above, you can refer to this document by Agilent Technologies: http://www.chem.agilent.com/Library/applications/5989-6068EN.pdf
Depending on the columns you have available and whether you wish to derivatise your samples, there are numerous articles employing phenylisothiocyanate as the derivatising agent and utilise a C18 column with a sodium acetate (and TEA) buffer system. An adaptation of this technique for free amino acids should be amenable to your analytes. I have used the following procedure with good success on amino acids:
Gheshlaghi, R., J.M. Scharer, M. Moo-Young, and P.L. Douglas, Application of statistical design for the optimization of amino acid separation by reverse-phase HPLC. Vol. 383. 2008. 93-102.
thanks for the document from Agilent you indicate. it completes weel the previsous answer of Niels. I now understand the role of TEA.
in fact, as my results demonstrated it, i do dot need to add TEA with the kinetex PFP column i use. On the contraty, TEA decreases the retention time of the analytes.
i also keep in mind the idea of derivatization, but i'm not generally prone to perform derivatization.
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