Fluorescence properties of carbazole-9-ylpropionic acid and its application to the determination of amines via HPLC with fluorescence detection
Fluorescence spectra of carbazole-9-ylpropionic acid (CRP), in the presence of different halide salts, organic solvents and surfactants and at various temperatures were investigated. The quenching constants of NaF, NaCl, NaBr, NaI and CH3I to CRP are 9.4, 12.6, 104, 456 and 560, respectively. Studies on the fluorescence spectra of CRP in 25 solvents indicated that the CRP fluorescence intensity increases with increasing polarizability of solvents and decreases on elevating the temperature. The influence of surfactants on the fluorescence spectra of CRP can be classified as two types: monomer quenching (below the CMC) and the micelle effect (above the CMC). The monomer quenching constant K for cetyltrimethylammonium bromide (CTMAB) to CRP is 1.8 × 103 l mol–1. The binding constants K of micelles for CTMAB and TX-100 to CRP are 2.6 × 103× 103
l mol–1, respectively. The fluorescence intensity and emission wavelength of CRP in the presence of the anionic surfactant SDS show no difference. As an application study the use of carbazole-9-ylpropionyl chloride (CRP-Cl) as derivatization reagent for the simultaneous separation of polyamines and amino acids with pre-column fluorescence derivatization via LC is described. The derivatization and chromatographic conditions were optimized on a reversed-phase C18 column using a binary gradient. Studies on derivatization conditions indicate that primary and secondary amines react very fast with CRP-Cl in alkaline solution to give the corresponding fluorescent derivatives, which exhibit excellent sensitivity and stability. This method, in conjunction with a multi-step gradient, offers a complete resolution of amino acid and polyamine derivatives. The separation of polyamines extracted from plant tissue takes less than 25 min. Excellent response linearity is demonstrated for amounts of polyamines injected in the range 50–250 pmol. The relative standard deviations (n = 6) at the picomole level are <5% and detection limits (signal-to-noise ratio = 3) are at the femto mole level.