Chromatography Forum

Does anyone know about the stability of derivatized amino acids using various derivatization reagents? I need something I can manually derivatize my samples with that is stable for 3-4 hours prior to separation by HPLC with fluorescence detection. Am mostly interested in quantifying glutamate.

I have read that deriv using OPA/mercaptoethanol is not that stable, but can be improved by substituting tert-butylthiol or sulfite or N-acetyl-cysetine. Would these last 4 hours or so? What about NDA, CBQCA or this new AQC reagent from Waters which is supposed to be stable for 1 week? Anyone ever tried these?

Thanks....

folive

I remember of a review article that had all the structures of amino acid derivatization agents as well as a comprehensive table of several characteristics such as stability, wavelengths, sensitivity, ease of use etc. Hopefully I still have it around at home so I'll have a look later, but if you are in a hurry, a search might reveal it...

Thanks for your help. If you come across that review article, please let me know.

This is a good review I found on stability of different OPA derivatization methods:

Molnar-Perl I. Derivatization and chromatographic behavior of the o-phthaldialdehyde amino acid derivatives obtained with various SH-group-containing additives. J Chromatogr A. 2001 Apr 13;913(1-2):283-302.

folive

Foline,

The review, "Electromigration methods for amino acids, biogenic amines and aromatic amines, J. Chromatogr. B 747, 2000, 1-19, has a table with all the derivatization agents structures (14 of them) and references to them... I couldn't find the review with the table summarization the characteristics of each reagent.

I wouldn't personally go with OPA if you care about derivative stability (Hans has some strong opinions about it...). I would probably go with the AQC reagent or maybe FMOC...

Yes, I wouldn´t touch it, unless someone could convince me with some unequivocal evidence.... Trimolecular reactions (or pseudo trimolecular) are often messy, even with stable reagents, products. Dansyl-Cl was also a pain in our hands. FMOC-Cl was useful, one can buy FMOC-aminoacid derivatives, which can be kept in the refrigerator almost indefinatly.

If you can not "escape" from fluorescence detection and stability is an issue, FMOC is "the method" for you.

If you can get an UV detector, I would recommend PITC.

Dear Folive,

I have used FMOC-Cl in many cases and always with good results. The stability of these derivatives is measured in hours or days (in solution). Also it can be easily done at the moment of analisis using automated injectors.

If you follow this way you have to take two things in mind, first is that the reaction is sensitive to pH conditions and it usually runs at pH>9, and second that there is the possibiloity of byproducts or multiproducts . If the compounds you derivatize contain more than one primary or secundary amine group you will have multiple signals. There is one byproduct that is always of some concern, FMOC-OH, is formed by FMOC-Cl reacting with water. This product however can be easily separated from the desired derivative.

There many examples of FMOC derivatization procedures in the literature.

Good Luck,

josebenjamin