Peptides, derivatization post-column

As already mentioned the EP wants to replace old TEC tests with separation methods of higher efficiency for example, the purity of amino acids is currently evaluated by a TEC test for ninhydrin-positive substances that is only able to find and limit amino acids to 0.5%. However, this test is only valid in the case the amino acids are produced by the cleavage of peptides/proteins and purification. The ninhydrin method is also used in the amino acid analysis of peptides, utilizing a cation-exchange chromatography with a post-column derivatization and a subsequent UVA is detection. This method is often used in industries for purity evaluation of amino acids. 

For analyses of synthetic peptides that are generally available in larger amounts than isolated natural peptides and proteins, the ninhydrin detection method is fully sufficient. The alternative detection method with orthophthaldialdehyde, which improves the sensitivity by a factor of five, was originally proposed for post-column derivatization, 34 but is presently used mainly in pre-column derivatization techniques. 35,36 ... 

Ye, M. L., Hu, S., Quigley, W. W. C., and Dovichi, N. J., Post-column fluorescence derivatization of proteins and peptides in capillary electrophoresis with a sheath flow reactor and 488 nm argon ion laser excitation, J. Chromatogr. A, 1022, 201, 2004. 

These approaches can be designated as post-column derivatization methods and can be performed with the appropriate devices. Due to the relatively slow reaction rates of many biochemical reactions, off-line detection may be preferable. In this case, incubation and reaction times can be adjusted independently of the separation speed. In Fig. 5, the separation of eight microcystins (cyclic peptides) and similar compounds is illustrated. With a group-selective antibody against microcystins, the respective toxin peaks can be unambiguously detected. Moreover, the ELISA detection is about 100 times more sensitive and much more selective than UV detection [18]. 

---