AN OXAZINE REAGENT FOR DERIVATIZATION OF CARBOXYLIC-ACID ANALYTES SUITABLE FOR LIQUID-CHROMATOGRAPHIC DETECTION USING VISIBLE DIODE LASER-INDUCED FLUORESCENCE

This study reports the use of Nile Blue, an oxazine dye, as a derivati zation reagent that fluoresces in the far-red spectral region and is s uitable for derivatization with carboxylic-acid-containing analytes. M odel carboxylic acid analytes such as benzoic acid, acetic acid, pheny lacetic acid and hexanoic acid have been reacted as acid chlorides to form Nile Blue derivatives. The synthesis product of the Nile Blue ben zoic acid derivative was confirmed using electrospray-mass spectrometr y, infrared spectrometry, H-1 and C-13 nuclear magnetic resonance, rev ersed phase liquid chromatography (RP-HPLC), normal phase-thin layer c hromatography, and spectral characterization. The synthesized Nile Blu e derivatives, separated from reaction by-products with RP-HPLC, all d emonstrated an approximately 10-fold drop in molar absorptivity and re lative quantum yield. In addition, a 40 nm increase in Stokes shift wa s observed. A portion of the fluorescence was regained through post-co lumn ionization of the Nile Blue benzoic acid derivative at pH 12. A R P-HPLC limit of detection of 88.25 fmol on column has been reported wi th conventional fluorescence detection-post-column ionization of the N ile Blue benzoic acid derivative. A limit of detection of 1.99 fmol on column (3.98 x 10(-11) M) has been demonstrated for the Nile Blue ben zoic acid derivative with the use of a laboratory-constructed visible diode laser fluorescence detector.