Sequential micellar electrokinetic chromatography analysis of racemization reaction of alanine enantiomers

A novel method for online monitoring racemization reaction of alanine (Ala) enantiomers was developed, by combining sequential sample injection and micellar electrokinetic chromatography (MEKC) technique. Various conditions were investigated to optimize the sequential injection, Ala derivatization and MEKC chiral separation of d-/l-Ala. High reproducibility of the sequential MEKC analysis was demonstrated by analyzing the standard Ala samples, with relative standard deviation values (n = 20) of 1.35%, 1.98%, and 1.09% for peak height, peak area and migration time, respectively. Ala racemization was automatically monitored every 40 s from the beginning to the end of the reaction, by simultaneous detection of the consumption of the substrate enantiomer and the formation of the product enantiomer. The Michaelis constants of the racemization reaction were obtained by the sequential MEKC method, and were in good agreement with those obtained by traditional off-line enzyme assay. Our study indicated that the present sequential MEKC method can perform fast, efficient, accurate and reproducible analysis of racemization reaction of amino acids, which is of great importance for the determination of the activity of racemase and thus understanding its metabolic functions.