By capillary gas chromatogra¬phy on a chiral stationary phase it is possible to separate all protein amino acids to¬gether with their enantiomers. Furthermore also nearly all non-proteinogenic α-amino acids and most of the N-methyl-amino acids can be analyzed in respect of their enantiomeric purity using this method. Amino acids with more than one chiral center are separated into all opti¬cal antipodes so as isoleucine, threonine or cystine. The amino acids must usually be deprotected, followed by ester¬ification and acylation using achiral reagents to obtain volatile derivatives for the chiral separation. The cleav¬age is performed either during esterification or in a separated hydrolysis step with aqueous hydrochloric acid. Fmoc cleavage is performed with piperidine at 0°C. The method of cleavage giving minimum racemisation is chosen. Often derivatives can be separated without cleavage (e.g. Acm, Boc, Bzl or Acetyl amino acids). If cleavage or hydrolysis is necessary, mass selective detection can increase the accuracy of the result for some derivatives.
The second method uses chiral columns for HPLC. A couple of derivatives can be separated on chiral HPLC columns without derivatization, if they have a chromophoric group like Fmoc. The lower selectivity of the chromatographic column is often compensate by the high selectivity of the detector.

HPLC and GC(-MS) results may differ if the sample is contaminated by any other derivative of the same amino acid with different enantiomeric purity. GC(-MS) gives as result the sum of the enantiomers of all these derivatives, HPLC analyzes the enantiomeric purity of the specified amino acid derivative only.

Results: Determination of the optical antipodes down to 0,1% (LOQ) .

• For description of the method download PDF.