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

A rapid and sensitive derivatization method, using carbazole-9-yl-acetyl ch loride (CRA-Cl) as derivatization reagent, for the simultaneous determinati on of amino acids and biogenic amines with pre-column fluorescence derivati zation via liquid chromatography is investigated. The wavelength of maximum emission for the CRA derivatives is 365 nm (lambda(ex) 335 nm). Fluorescen ce spectra of non-acyl chloride CRA in the presence of various salts, organ ic halides and different surfactants an also studied. The quenching constan ts of NaF, NaCl, NaBr, NaI, and CH3I to CRA are 9.4, 12.6, 40.4, 56.2, and 102 M-1, respectively Heavy atoms (above 1.0 mu M) will seriously interfere with CRA emission spectra tested in the presence of halogen-salts and iodo methane. On the basis of Stern-Volmer equation for static quenching and a s olute-micelle interaction linear relationship, the monomer quenching consta nt K of hexadecyltrimethylammonium bromide (CTMAB) to CRA is 3.0x10(3) M-1. The binding constants K of micelle of CTMAB and TX-100 to CRA are 5.4x10(3 ) M-1 and 2.0x10(3) M-1, respectively. in addition, in a kinetic analysis o f CRA emission intensity (In I-em) a linear correlation between ln I-em and 1/T was found. The slope of the working curve yields an emission stabiliza tion energy of 17.8 kJ/mol. Derivatization and chromatographic conditions f or simultaneous separation of derivatized amino acids and biogenic amines h ave also been optimized on a reversed-phase C-18 column using a binary grad ient. Studies on derivatization conditions indicate that primary and second ary amines react very fast with CRA-Cl in alkaline solution to give the cor responding fluorescent derivatives, which exhibit excellent sensitivity and stability. This method, in conjunction with a multi-gradient program, offe rs a complete resolution for amino acids and biogenic amines. For rapid sep aration of biogenic amines in plant tissue, an isocratic elution is used. E xcellent 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 (signa l-to-noise ratio=3) are levels of fmol. (C) 1999 Elsevier Science B.V. All rights reserved.