On-the-Fly Fluorescence Lifetime Detection in Liquid Chromatography with Data Collected Simultaneously at Multiple Emission Wavelengths

Abstract

A novel liquid chromatography (LC) fluorescence detector simultaneously generates fluorescence decay curves at multiple emission wavelengths. The fourth harmonic (266 nm) excitation light from a pulsed Nd:YAG laser is focused into a standard LC flow cell. A small spectrograph disperses the fluorescence. Fiber-optic delay lines positioned in the exit focal plane time the arrival of photons at the photomultiplier tube detector. Four wavelength channels with 50 ns channel spacing were used in this study. The intensity-decay time waveform is averaged with a digital storage oscilloscope. Temporal overlap between adjacent wavelength channels is removed during the post-processing of the data. System performance was characterized for chromatographic elution of polycyclic aromatic hydrocarbons (PAHs). Detection limits are 1.6 ppb for naphthalene and 0.14 ppb for fluorene. Resolution of co-eluting 1,4-dimethyl-naphthalene and anthracene components on the basis of differences in their lifetimes and spectral distributions is demonstrated.

Use of ultrabright LEDs for the determination of static and time-resolved florescence information of liquid and solid crude oil samples

“…One examples in the field of PAHs is Gooijer et al [2] where a quadrupled Nd-YAG laser (266 nm) is used to study PAH metabolites in biota with LIF detection in capillary electrophoresis. A similar set-up has also been reported by Burt et al [3], Quinn et al [4], and in a more mobile version by Karlitschek et al [5]. As the fluorescence intensity is very sensitive to the sample preparation and media effects, there have been recent attempts to investigate reference materials; see Ref.…”

Label‐free analysis in chip electrophoresis applying deep UV fluorescence lifetime detection

“…An even more attractive feature of a fluorescence lifetime measurement is the possibility to distinguish between different analytes [31][32][33][34], which can also serve to identify co-eluting peaks [32][33][34][35][36]. Even for analytes that contain identical UV fluorophores, e.g.…”

“…The group also presented lifetime detection of fluorescent dyes in CE [43]. Gillispie and co-workers reported on on-the-fly fluorescence lifetime determination of PAHs in HPLC with excitation at 266 nm and demonstrated deconvolution of co-eluting peaks [34,36]. Furthermore, He and Geng reported on fluorescence lifetime detection via phase modulation at 488 nm excitation in CE [44], while Soper's group utilized near IR excitation for TCSPC analysis of labeled oligonucleotides separated by CE [33] as well as by MCE [45].…”

Electronic spectral detection in liquid chromatography

“…Burt, Dvorak, Gillespie, and Oswald [45] coupled an HPLC to detect and identify several PAHs through measurement of their fluorescence decay lifetimes. Several wavelengths were monitored simultaneously to differentiate closely eluting peaks.…”