Analysis of multicomponent mixture and simultaneous enantioresolution of proteinogenic and non-proteinogenic amino acids by reversed-phase high-performance liquid chromatography using chiral variants of Sanger's reagent

Ravi Bhushan, Rajender Kumar

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35 Scopus citations

Abstract

Four chiral derivatizing reagents (CDR 1-4), namely, FDNP-l-Ala, FDNP-l-Val, FDNP-l-Phe, and FDNP-l-Leu, were synthesized using microwave (MW) irradiation by substituting one of the fluorine atoms in difluoro dinitro benzene (DFDNB) with l-Ala, l-Val, l-Phe, and l-Leu (CDR 1-4). The other set of CDRs, namely, FDNP-l-Phe-NH2, FDNP-l-Val-NH2, and FDNP-l-Leu-NH2, was also prepared. These reagents were used for synthesis of diastereomers of 18 proteinogenic and 08 non-proteinogenic amino acids, which were resolved by reversed-phase high-performance liquid chromatography using C18 column and gradient eluting mixture of aq.TFA and acetonitrile with UV detection at 340 nm. The reagents were used for resolution of a complex mixture of 18 racemic proteinogenic amino acids in a single chromatographic run of 65 min and to determine concentration of the d-amino acid in a solution of dl-amino acid. The resolution (R S) and selectivity (α) obtained for the two sets of diastereomers were compared among themselves and among the two groups. The method was validated for accuracy, precision, limit of detection (LOD), and limit of quantification. LOD is 0.001% impurity of d-enantiomer.

Original languageEnglish
Pages (from-to)1697-1705
Number of pages9
JournalAnalytical and Bioanalytical Chemistry
Volume394
Issue number6
DOIs
StatePublished - Jul 2009
Externally publishedYes

Bibliographical note

Funding Information:
Acknowledgment The authors are thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India for award of a junior research fellowship to (R.K.) and to the Alexander von Humboldt Foundation, Bonn, Germany, for donating Knauer HPLC equipment (to RB in 2006).

Keywords

  • Chiral separation
  • Chiral variants of Sanger's reagent
  • Liquid chromatography
  • α-Amino acids

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