Chromatography Forum

Folks,
I am hoping someone can help me out by forwarding a method for the determination of purity for amino acid residues by LC. I am currently roadtrialing a method written by an LC vendor using OPA as a derivatising agent. However, the method requires precolumn derivatisation and as described by the procedure, this step is automated using the autosampler. However, I am attempting to do this manually prior to injection however, I am not getting repeatable results. This is partly due (I suspect) to the instability of the OPA derivative.
Are there other established methods which can be optimised to determine purity of individual amino acid residues? I have read CE is another option but how robust is this? Does anyone know if titrimetry is another option?
Regards
Colette

Colette,

Accurate amino acid analysis is not a trivial matter as you have discovered. There are several options other than OPA for derivatization and analysis. The most common are PITC, FMOC, and dansyl chemistries. Check out the following website for more information.

http://www.abrf.org/index.cfm/group.sho ... sis.19.htm

Also check Beckman and Waters, both of which have specific applications for reversed-phase analysis of derivatized amino acids. There are advantages and drawbacks to each of the methods. Good luck!