Improved Chiral Separations for Enantiopure D- and L-Amino Acids

June 1, 2016

Several D-amino acids have been found to be important in brain neurochemistry and changes in the levels of these amino acids coincide with development of different diseases. For example, the D-form of serine is involved in modulation of the NMDA receptor and has been implicated in a broad spectrum of disorders including schizophrenia, ischemia, and epilepsy.

D-amino acids have historically been considered unnatural since naturally produced proteins are composed exclusively of L-amino acids. While bacterial cell walls were known to contain D-amino acids, their presence in humans and higher organisms has been reported more recently.

Crown-ether chiral stationary phases (CSPs) have been found to be especially well suited for separation of D- and L-amino acid enantiomers. Regis Technologies' crown-ether CSP ChiroSil® is a trifunctionally bonded (18-crown-6)-2,3,11,12-tetracarboxylic CSP available in both (+) and (-) conformations which thereby enables inversion of the elution order of the D- and L- isomers.


Figure 1: HPLC analytical screen of Serine. Column: ChiroSil® SCA(-), 15 cm × 4.6 mm, 5 µm. Mobile Phase: 84% MeOH/16%H2O, 5 mM HClO4. k'1: 1.37 α: 1.99

In this application note, we present separation of the D- and L-isomers of two neurophysiologically relevant amino acids, serine and glutamic acid, by high pressure liquid chromatography (HPLC) using the CSP.


Figure 2: HPLC analytical screen of glutamic acid. Column: ChiroSil® SCA(-), 15 cm × 4.6 mm, 5 µm. Mobile phase: 84% MeOH/16%H2O, 5 mM HClO4. k'1: 1.45 α: 1.60.

Experimental Conditions

Compounds were separated on ChiroSil® SCA(-) chiral columns using standard dimensions, 15 cm × 4.6 mm i.d. packed with 5 micron particles. ChiroSil® is a registered trademark of RStech Corporation of Daejeon, South Korea.

Conclusions

Baseline enantiomeric resolution of serine and glutamic acid amino acids was achieved in less than 10 min using the crown-ether CSP method described here. The ChiroSil® CSP is a beneficial column in analyzing amino acids in the neurochemistry field.

Regis Technologies, Inc.
8210 Austin Avenue, Morton Grove, IL 60053
tel. (847) 967-6000, fax (847) 967-5876
Website: www.registech.com