1.2.5. HPLC of amino acids as dansyl and dabsyl derivatives

Abstract

Summary Pre-column derivatization of amino acids with 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) or 4-dimethylaminoazobenzene-4′-sulfonyl chloride (dabsyl chloride) is described. These pre-column derivatization methods enable a sensitive HPLC analysis of amino acids. Dansyl derivatization has originally been applied to the sequential analysis of peptides and proteins. It finds another use in biochemistry for the fluorogenic labeling of proteins and enzymes. Dansyl chloride is the most widely used for the derivatization of amino acids. Dansyl chloride readily reacts with primary and secondary amino groups of amino acids. The reaction medium is usually an aqueous-organic mixture (e.g., 1:1 acetone-water) adjusted to a pH of 9.5-10. Dansylation reaction is usually carried out at elevated temperatures. Various dansylation reaction conditions are reported, involving 60 °C for 60 min or at 38 °C for 90-120 min. Dansyl amino acids absorb light in the UV region. For example, absorption maxima are observed at 214, 246 and 325 nm for dansyl glycine, and the absorption at 214 nm is the strongest. Dansyl group is also fluorescent and dansyl amino acids can therefore be detected by a fluorimetric detector. The excitation and emission wavelengths for dansyl glycine are 324 and 559 nm, respectively. Dansyl amino acids are mostly separated in the reversed-phase mode on a C8 or C18 column with a linear gradient. Dabsyl chloride has a number of advantages over other derivatization methods, including a simple derivatization procedure, very good stability, good reproducibility and a good limit of detection for the method, complete HPLC separation of all the amino acids, and specific detection at a wavelength in the visible region. Dabsyl chloride also reacts with primary and secondary amino groups of amino acids as does dansyl chloride. Dabsylation reagent solution can be prepared by dissolving dabsyl chloride in acetone or in acetonitrile, followed by mixing with buffer (e.g., carbonate, pH 8.5-9.5). Derivatization of amino acid with dabsyl chloride is also carried out at elevated temperatures as for dansylation. For example, samples were incubated at 70 °C for 15-30 min. There have been a number of literatures dealing with application of dansylation to the determination of amino acids contained in various sample sources, involving biological fluids, tissues, foods, peptide or protein hydrolyzates, etc. Dabsylation also covers nearly the same application areas as for dansylation. Enantiomeric separation of dansyl and dabsyl amino acids can be carried out by ligand-exchange chromatography. The use of native, and derivatized β- and γ-cyclodextrin stationary phases as well as the use of γ-cyclodextrin mobile phase additive are other options for the chiral separation of these derivatized amino acids.