Construction of a light‐emitting diode fluorescence detector for high‐performance liquid chromatography and its application to fluorometric determination of l‐3‐hydroxybutyrate

Abstract

This study employed a new light source, a light-emitting diode (LED), for fluorescence detection of high-performance liquid chromatography to measure the concentration of trace constituents in biological fluids. Using l-3-hydroxybutyrate ( l-3HB) as a tested trace compound, the function of the new system was compared with that of the current commercially available model. A detailed schematic diagram of the path of the detection rays in the LED detector is given. A voltage-stabilizer for the drive circuit was designed with an input of 10 V and an output of 8 V, and another voltage regulator was used to maintain a constant 8 V. Then the regulator was used to set the output voltage for the LED at 2.8 V by two external resistors. Replacing the xenon lamp with LED, this system provided higher photon density and a narrow spectrum at a wavelength of 491 nm. At room temperature (22.1°C), the average temperature of six places in the chamber of LED detector was 22.1°C compared with 51.1°C in the xenon detector. The spectra of the excitation light sources were measured. Compared with the xenon lamp, approximately 1.32 times higher excitation intensity was obtained by the LED source. The accuracy of detection of l-3HB in 50 μL of rat serum was 99.85-100.85%, and the intra-day and inter-day precision values were within 8.99 and 13.90%, respectively. The limit of detection of l-3HB was approximately 0.73 µM (signal-to-noise ratio 3). The sensitivity of the proposed LED detector was comparable to that of traditional fluorescence detectors using xenon arc lamps; however, the cost and operating temperature of LED lamps were far lower. This assay system could be further used to detect trace constituents in various samples.