Fluorescence Measurements^[1]
Fluorescence spectra are recorded on a Hitachi MPF-24 Fluorescence Spectrophotometer. The emission spectrum is determined at the optimal wavelength of 464 nm. Thin-layer chromatography is carried out on Brinkmann glass supported analytical silica G thin-layer plates without fluorescent indicator. The NBD derivatives are visualized on thin-layer plates either by examination under an ultraviolet lamp with a long wavelength filter or by using the same lamp with a 2×2-in. Corning 5-57 filter and viewing the spots through a 2×2-in. Corning 3-68 filter. The former method produces a more intense fluorescence, but the latter produces a much greater contrast appearing as white spots against a dark background. NBD-Cl (NBD chloride) is allowed to react with amino acids and peptides under the conditions. Into 3 mL Pyrex test tubes are pipette the following: (1) 2.0 mL of ethanol, (2) 0.1 mL NBD-Cl solution in ethanol 1.41 pg/mL, (3) 0.1 mL ethanol saturated with sodium acetate, (4) 0.1 mL of amino acid solution 400-600μg/mL, and the mixture incubated at 75°C for 20 min. For reaction with peptides, 1.5 mL of ethanol, 0.1 mL ethanolic sodium acetate, 0.4 mL ethanolic NBD-Cl, 1.41 μg/mL, are added to 0.4 ml of the peptide and incubated under the same conditions.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Fager RS, et al. The use of NBD chloride (7 chloro-4-nitrobenzo-2-oxa-1,3-diazole) in detecting amino acids and as an N-terminal reagent. Anal Biochem. 1973 May;53(1):290-4.  [Content Brief]