Development of Pirkle-Type Chiral Stationary Phases for the Enantioselective and Comprehensive HPLC Analysis of All Proteinogenic Amino Acids

Abstract

The Pirkle-type chiral stationary phases for enantioselective high-performance liquid chromatographic (HPLC) separation of all the proteinogenic amino acids plus allo-forms (allo-isoleucine and allo-threonine) were designed and developed. The developed three columns having N-(3,5-dinitrophenylaminocarbonyl)-l-amino acids at their chiral recognition sites successfully separated the enantiomers of all the proteinogenic amino acids and allo-forms individually. The obtained separation factors of the enantiomers were 1.08–1.42, 1.09–1.52 and 1.14–1.55 for the Singularity CSP-001S, 016S and 017S columns, respectively. The elution orders of the enantiomers were ideal for all the amino acids using these three columns; d-enantiomers (normally minor isomers in biological matrices) eluted faster than the predominant l-forms indicating that these columns are helpful for the determination of trace amounts of the d-forms. The separation profiles of 3 chiral amino acids (alanine and 2 model interfering amino acids) revealed that these newly designed columns have different separation properties. The accurate analysis of trace amounts of d-amino acids in complicated biological samples is often disturbed by the co-elution of numerous interfering substances. Therefore, the newly designed stationary phases would contribute to cross-checking the quantitative values of chiral amino acids besides their comprehensive determination in real-world matrices.