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The Application Notebook
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 ChiroSilÂ® 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: ChiroSilÂ® SCA(-), 15 cm Ã 4.6 mm, 5 Âµm. Mobile Phase: 84% MeOH/16%H2O, 5 mM HClO4. k'1: 1.37 Î±: 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: ChiroSilÂ® SCA(-), 15 cm Ã 4.6 mm, 5 Âµm. Mobile phase: 84% MeOH/16%H2O, 5 mM HClO4. k'1: 1.45 Î±: 1.60.
Compounds were separated on ChiroSilÂ® SCA(-) chiral columns using standard dimensions, 15 cm Ã 4.6 mm i.d. packed with 5 micron particles. ChiroSilÂ® is a registered trademark of RStech Corporation of Daejeon, South Korea.
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 ChiroSilÂ® CSP is a beneficial column in analyzing amino acids in the neurochemistry field.
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