1.1.3. Chiral separation of amino acids by gas chromatography

Abstract

The chirality of amino acids plays an important role in many fields of biosciences including synthetic peptide chemistry and peptide drug design. Analytical methods are required to assure their optical configuration in either their free state or peptide bonded form. Besides established enzymic and a multitude of advanced liquid chromatographic approaches, gas chromatographic procedures are still attractive. This is attributed to their capability to resolve complex mixtures of amino acid enantiomers after suitable derivatization, and to determine enantiomeric ratios with high sensitivity and reliability using GC-MS in the selected ion monitoring mode. Whereas the indirect determination of amino acids by forming diastereomeric using chiral derivatizing reagents has now limited applications (but does not need a special column), direct enantioseparation on chiral stationary phases is widely used. Among the numerous chiral phases described in the literature only Chirasil ® -Val, representing dimethylpolysiloxane bonded valine tert butylamide (in either the L- or the D-form) and, more recently, functionalized cyclodextrins are commercially available for routine applications. The deuterium labeling method together with GC-MS enables the estimation of the amount of racemization of amino acids caused by total hydrolysis in peptides and proteins. Limitations of the gas chromatographic approaches, however, are the need for derivatization of amino acids in order to generate volatile derivatives, problems encountered with the basic amino acids and amino acid amides, and limited stability of derivatives of hydroxy amino acids. Abbreviations: common amino acids are abbreviated to the three letter nomenclature; special amino acids are abbreviated as follows: Nle, norleucine; Dap, 2,4-diaminopimelic acid; Hyl, hydroxylysine; Hyp, hydroxyproline; Gaba, ?-aminobutyric acid Iva, isovaline or 2-ethylalanine; TFA, trifluoroacetic acid; TFAA, trifluoroacetic acid anhydride; PFPA, pentafluoropropionic acid; PFPAA, pentafluoropropionic acid anhydride; Me, methyl; Prp, propyl, DCM, dichlormethane; BHT, tert. Butylhydroxy toluene; GC, gas chromatography; MS, mass spectrometry; SIM, selected ion monitoring