Enantioselective separation of unusual amino acids by high performance liquid chromatography

• Separation of unusual AAs enantiomers was performed in HPLC.
• Teicoplanin or polysaccharide based chiral stationary phases were employed.
• Non-blocked AAs enantiomers were best separated on teicoplanin based columns.
• Blocked AAs enantiomers were best resolved on Chirobiotic T or Chiralpak IC columns.
• Chiral selector structure affected elution order of enantiomers.

Unusual amino acids, i.e. amino acids not encoded by DNA, play fundamental roles in many scientific fields. Since the single enantiomeric form can cause different and often serious response of organisms, chiral separations of unusual amino acids are irreplaceable tools in their study and their employment. Two types of chiral stationary phases, two teicoplanin-based and four polysaccharide-based columns, were used. Separation conditions of reversed phase mode, polar organic mode and hydrophilic interaction chromatography were evaluated and compared. All columns used exhibited significantly different enantioselectivities. Teicoplanin-based chiral stationary phases, especially Chirobiotic T column, were able to separate almost all enantiomers tested, with the exception of Z-D,L-4-F-phenylalanine ethyl ester. No partial enantioseparation of this analyte was obtained on teicoplanin-based chiral stationary phases, while baseline enantioresolution was achieved on polysaccharide-based columns. Change of elution order of L- and D-enantiomers was proved regarding the chiral stationary phase used.