Liquid Chromatography Detectors - The Fluorescence Detector > The Multi Wavelength Fluorescence Detector > Page 60

The instrument is quite complex but is extremely versatile. The use of the detector to optimize both the excitation light and the fluorescence light to provide high selectivity for the Fluoropa derivative of neomycin is shown in figure 39. It is a very good example of the selection of a specific excitation light wavelength and the complementary emission light wavelength to provide maximum sensitivity.


Column: Supercosil LC–8, 15 cm x 4.6 mm, 5 mm particles: Mobile Phase: tetrahydrofuran : 0.0056M sodium sulfate/0.007M acetic acid/0.01M pentane sulfonate, 3:97. Flow rate: 1.75 ml/min. Post Column reagent: 1L 0.4M boric acid/0.38M potassium hydroxide containing 6 ml 40% Brij–35, 4 ml mercaptoethanol, 0.8g o-phthalaldehyde. Flow rate 0.4 ml/min. Mixer 5 cm x 4.6 mm column packed with glass beads. Reactor 10 ft x 0.5 mm knitted Teflon capillary tubing. Reaction Temperature 40oC. Sample 20 ml of a mobile phase extract of a commercial sample. Excitation wavelength 365 nm;emission wavelength 418 nm.


Courtesy of Supelco Inc.

Figure 39. Detection of Neomycin OPA Derivative at an Excitation Wavelength of 365 nm and an Emission Wavelength of 418 nm