Chromatography Forum

Hi,
I am trying to develop a HPLC method for purity analysis of an aminoacid with a tertiary amine group and no chromophores (like dimethyl beta - alanine).

The target impurities are also amino acids with a tertiary amine group.

I don´t have ELSD or MS detector. I only have UV detectors.

Inicially I tried to work at 190-200 nm but the sensibility (as expected) is low in order to search impurities at 0.1 % level.

So I am looking a simple pre column derivatization method, but I have a problem: most of the currently used methods for aa derivatization involve the use of amine derivatizating agents that are only apropiate for aa that contain primary or secondary amine groups.

I need your advice on derivatization techniques for the carboxylic acid moiety that are usufull for HPLC UV detection.

Maybe you can recommmend a suitable approach to tackle the problem.

Thanks in advance for your thoughts.

Ester formation with an alkohol that contains a sufficiently visible tag? For example, benzylalkohol.

Well, I personally have tried only EDC (which is carbodiimide) from PIERCE to modify protein carboxyls (see http://www.piercenet.com/files/1601361Crosslink.pdf for more information). Also there should be some diazoalkanes available to do the desired modifications (diazomethan, if I recall correctly, is used in GC to derivatize carboxyls; but I might be wrong, 'cause have never done it myself). Check with Molecular Probes ( http://probes.invitrogen.com/handbook/s ... /0303.html ) - very nice book, plus a lot of references.

Good luck.

Serg.

Thank you Uwe.

Serg many thanks for your references, they contain a lot of information.

¿Any more suggestions?

There are a couple of textbooks that deal with derivatization of functional groups, with experimental details.

1) "Handbook of derivatization for chromatography" K. Blau & G. King

2) "Title similar to above" D. R. Knapp et al.

Please update the forum when you have a successful method established,

JMB