A Method to Study Column Efficiency in Indirect HPLC Enantioseparation

Abstract

Four ways are proposed for the comparison of true column performance for various C₁₈ columns: plotting the resolution vs. the retention factor, plotting the resolution rate vs. retention factor, calculating the retention time needed for a given resolution, or the resolution obtained in a given time. The utilization of these methods is shown for eleven C₁₈ columns in the separation of the enantiomers of erythro‐β‐methyl amino acids. The indirect chromatographic method involved pre‐column derivatization with new chiral derivatizing agents (CDAs), such as (1S,2S)‐1,3‐diacetoxy‐1‐(4‐nitrophenyl)‐2‐propyl isothiocyanate [(S,S)‐DANI], and (S)‐N‐(4‐nitrophenoxycarbonyl)phenylalanine methoxyethyl ester [(S)‐NIFE], and the separation of the diastereomers formed on different types of C₁₈ columns. The different columns were compared in systematic chromatographic examinations. The influence of various parameters of the columns on the resolution and the time of analysis are discussed.

Keywords:

• High performance liquid chromatography
• Column efficiency
• erythro‐β‐Methyl amino acids
• (1S,2S)‐1,3‐Diacetoxy‐1‐(4‐nitrophenyl)‐2‐propyl isothiocyanate
• (S,S)‐DANI
• (S)‐N‐(4‐Nitrophenoxycarbonyl)phenylalanine methoxyethyl ester
• (S,S)‐NIFE

Acknowledgment

This work was supported by OTKA grant T 042451.