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Enantiomeric Separation & Analysis of Native or Derivatized Amino Acids using Chiral HPLC and Mass Spectrometry

By: Ric Cone and Wayne K. Way, Reporter US Edition 25.1

Ric Cone and Wayne K. Way

Separating native and derivatized amino acid enantiomers or diastereomers using chiral HPLC is integral to research in the pharmaceutical, bioscience, medical diagnostic, food, and agricultural sectors. Amino acids contain an asymmetric center, and determining the relative amounts of D- and L-amino acids is essential for natural product characterization, certain genetic disease diagnosis, and solid phase peptide synthesis.

When achiral phases are used for chiral separation, derivatization with reagents is required. Purity of the reagents, derivative instability, and long prep time remain limitations when compared with use of chiral stationary phases (CSPs).

Astec, a leading name in chiral chromatography, is now part of Supelco and the Sigma-Aldrich Family, and offers a wide variety of both silica-based and polymeric chiral HPLC columns. Astec’s offerings for analysis of natural and derivatized amino acids include, CHIROBIOTIC, CYCLOBOND, P-CAP, and Astec CLC columns. The CHIROBIOTIC T stationary phase is an amphoteric glycoprotein teicoplanin, stably bonded to 5 μm, high purity, spherical silica gel. CHIROBIOTIC T will separate all amino acids in reversed phase mode, and can replace crown ether or ligand exchange columns. It displays unusual behavior by giving better resolution and longer retention with increasing organic modifier. Amino acids can be separated in reversed phase mode using a buffer to lower the pH and increase retention. Amino acid derivatives such as methyl esters and peptides may be separated in a polar organic mode akin to the cyclodextrin technique except the typical mobile phase consists of 46:54:2:2, ACN/MeOH/HOAc/TEA. Normal phase separations are also possible with hexane/ethanol mobile phases.

Table 1 describes the best starting mobile phase compositions for the separation of natural amino acids and their Nblocked derivatives. All CHIROBIOTIC columns can be used with the widest variety of MS compatible solvent systems.

In addition to their use for amino acid analysis, CHIROBIOTIC stationary phases have demonstrated versatility in both reversed phase and normal phase solvents, and in the polar organic and polar ionic modes.

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