Practical Protein Chemistry - A. Darbre 1989

Enantiomeric analysis of amino acid mixtures by high-performance liquid chromatography
Conclusions

Any laboratory equipped with a high-performance chromatograph and an appropriate detector can easily set up a two-stage chromatographic system for the enantiomeric Separation of Amino Acids. Standard commercial columns can be used without modification. Separation efficiency can be improved by establishing an acetonitrile concentration gradient, which allows the simultaneous analysis of neutral and aromatic amino acids, and by installing a valve at the Column outlet that diverts the bulk of the eluate to a fraction collector and only a small portion to the detector for quantification.

In an alternative approach to Enantiomeric analysis, individual Amino acids are collected separately from a cation-exchange resin using a volatile buffer (0.1 M pyridine-acetate, pH 3.1; followed by 0.4 M pyridine-acetate, pH 5.3), followed by reversed-phase isomer separation using a chiral additive (Engel, 1981, private communication). In cases where the mixture does not contain all protein amino acids, the Ion-exchange Chromatography step may be omitted.

Modifying the configuration of the chiral additive component alters the elution order of the D- and L-isomers from the column; the results of such an experiment can serve as additional confirmation of their ratio. A shift in this ratio indicates the presence of impurities in the sample.