Analysis of the enzymatic racemization of d-aspartic acid to l-aspartic acid by the on-line coupling of a solid-phase extraction column and a ligand-exchange high-performance liquid chromatography column

Abstract

d-Aspartic acid can be enzymatically biotransformed with d-amino acid oxidase and aminotransferase to l-aspartic acid. The reaction was surveyed at three temperatures and a period of 3 days, however, l-aspartic acid can be produced only at the reaction temperature 90°C. However, the separation of d-aspartic acid and l-aspartic acid by ligand-exchange chromatography showed matrix interference. Therefore, the column-switching technique by coupling a solid-phase extraction (SPE) column to the analytical ligand-exchange HPLC column was used to eliminate the matrix effect. The pretreatment of reaction samples with the SPE column was considered as a combination of size-exclusion chromatography and ion-pair chromatography. The ion-pair reagent was 0.005 M sodium 1-octanesulfonate aqueous solution adjusted to pH 2.2. Part of the first eluted peak from the SPE column was then switched through the ligand-exchange column and analyzed with a 0.25 m M Cu 2+ aqueous mobile phase of pH 3.6. The quantitative analysis of d- and l-aspartic acids was performed by the standard addition method. Overall, the separation and analysis of d- and l-aspartic acids in the enzymic solution was convenient, fast, and successful with the developed on-line LC–LC column-coupling and column-switching system.