Fluorescence properties of carbazole-9-yl-acetyl chloride and its application for the simultaneous determination of amino acids and biogenic amines via liquid chromatography with fluorescence detection

Abstract

A rapid and sensitive derivatization method, using carbazole-9-yl-acetyl chloride (CRA-Cl) as derivatization reagent, for the simultaneous determination of amino acids and biogenic amines with pre-column fluorescence derivatization via liquid chromatography is investigated. The wavelength of maximum emission for the CRA derivatives is 365 nm ( λ ex 335 nm). Fluorescence spectra of non-acyl chloride CRA in the presence of various salts, organic halides and different surfactants are also studied. The quenching constants of NaF, NaCl, NaBr, NaI, and CH 3I to CRA are 9.4, 12.6, 40.4, 56.2, and 102 M −1, respectively. Heavy atoms (above 1.0 μM) will seriously interfere with CRA emission spectra tested in the presence of halogen-salts and iodomethane. On the basis of Stern–Volmer equation for static quenching and a solute–micelle interaction linear relationship, the monomer quenching constant K of hexadecyltrimethylammonium bromide (CTMAB) to CRA is 3.0×10 3 M −1. The binding constants K of micelle of CTMAB and TX-100 to CRA are 5.4×10 3 M −1 and 2.0×10 3 M −1, respectively. In addition, in a kinetic analysis of CRA emission intensity (ln I em) a linear correlation between ln I em and 1/ T was found. The slope of the working curve yields an emission stabilization energy of 17.8 kJ/mol. Derivatization and chromatographic conditions for simultaneous separation of derivatized amino acids and biogenic amines have also been optimized on a reversed-phase C 18 column using a binary gradient. Studies on derivatization conditions indicate that primary and secondary amines react very fast with CRA-Cl in alkaline solution to give the corresponding fluorescent derivatives, which exhibit excellent sensitivity and stability. This method, in conjunction with a multi-gradient program, offers a complete resolution for amino acids and biogenic amines. For rapid separation of biogenic amines in plant tissue, an isocratic elution is used. Excellent response linearity is demonstrated over the injected amounts range 50–250 pmol for biogenic amines. The relative standard deviations ( n=6) at an analytical concentration of pmol level are <5%. Detection limits (signal-to-noise ratio=3) are levels of fmol.