Practical Protein Chemistry - A. Darbre 1989

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

S. WEINSTEIN (Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot, Israel), M. H. ENGEL (School of Geology and Geophysics, The University of Oklahoma, Norman, OK 73019, U.S.A.), P. E. HARE (Geophysical Laboratory, Carnegie Institution of Washington, D.C., U.S.A.)

The chromatographic Separation of amino acid optical isomers using high-resolution Methods is of considerable interest across many fields of research. This area of Enantiomeric analysis evolved simultaneously across several laboratories alongside advancements in High-Performance Liquid Chromatography (HPLC). It was initially proposed to separate amino acid enantiomers using polystyrene |2| and polyacrylamide |6| matrices with covalently attached chiral ligands capable of forming complexes with divalent copper ions. The same principle—Metal Ions complexed with chiral ligands, but in the Mobile phase—was later applied in reversed-phase chromatography [4]. An efficient and highly sensitive method was described for analyzing amino acid isomers using an aqueous solution containing a Cu (II)-L-Proline complex as the mobile phase, and a cation-exchange resin or reversed-phase silica gel as the stationary phase. However, this approach failed to achieve complete baseline separation for all amino acid enantiomers [3, 5], which was subsequently accomplished using their dansyl derivatives in a dual-Column system [8].

Further progress in this field is associated with the synthesis of Serine N,N-dialkylamino acids and their application, in the form of complexes with Cu (II) ions, as chiral stationary phases [9, 10]. In the methodology outlined below, RP-HPLC utilizing a chiral copper(II) N,N-di-n-propyl-L-alaninate complex (Cu-DPA) successfully resolved all protein Amino Acids into their respective enantiomers. To improve resolution, chromatography is performed in a two-stage process. In The First stage, The amino acid mixture is pre-fractionated into groups using a cation-exchange column under conditions similar to those used in conventional amino acid analyzers, but employing volatile buffers. Following solvent evaporation, each fraction is then subjected to enantiomeric analysis on an RP column with a chiral mobile phase. Post-column derivatization of the eluting components with o-phthalaldehyde in the presence of mercaptoethanol (OPA reagent) enables the fluorescence-based detection of sub-nanomole quantities of amino acids. Proline does not react with OPA and is instead identified via the ninhydrin reaction [7]. The analysis of Cysteine and cystine is performed following their oxidation to cysteic acid, which is well resolved from Other Amino Acids under the described conditions.