On-line fluorescence lifetime detection for chromatographic peak resolution

Time‐Resolved Fluorescence HPLC Detection Using Semiconductor Light Sources: Principles and Applications

“…This procedure gives rise to two effects: firstly, the lifetime around the background value can clearly be identified by the low noise, and secondly, the peaks in the chromatogram appear broader compared to the corresponding intensity peaks. The latter effect has already been reported in [6]. Additionally, the peaks are not perfectly rectangular due to an overlay of background signal and sample detection in the rising and falling edge of the peak.…”

“…Attempts to use time-resolved fluorescence techniques together with HPLC are not new. Previous investigations used commercial phase fluorometry (48000s phase-modulation spectrofluorometer from SLM Instruments, Inc., Urbana, IL, USA) with 450-W Xe-lamp [6] or an externally modulated HeCd-laser (325 nm) applying an external Pockels Cell [7]. The latter one describes a setup that was able to measure at different frequencies during a single chromatogram resolving two components in the sample at the same retention time.…”

“…In a later paper [8], the same group analyzed more rigorously the LoD for benzo[k]-fluoranthene and benzo[f]-fluoranthene with the same setup from reference [7] and found 1.6 ng and 0.08 ng, respectively. A modulated Xe lamp at 30.0 MHz with a setup similar to [6] was also used to investigate photomultiplier (PMT) baseline effects on modulated fluorescence data in HPLC [8]. In order to increase the sensitivity, a pulsed laser at 266 nm, i.e., a frequency-quadrupled Nd:YAG laser, with PMT detector and digital storage oscilloscope as the recording system was used.…”

Proof-of-concept analytical instrument for label-free optical deconvolution of protein species in a mixture

“…This technique has not been previously combined with liquid chromatography for the real-time detection of proteins, although there is precedent for distinguishing polycyclic aromatic hydrocarbon compounds (PAHs) that co-elute by HPLC, based on their fluorescence lifetimes. [13][14][15][16][17] However, the analytical instrumentation used to measure the lifetimes of PAHs is ineffective for distinguishing proteins because the difference in the fluorescence decay times between similar proteins can be less than 0.1 ns, which is a major challenge to resolve in real time. In addition, a shift to high-bandwidth detection electronics is required because proteins have fluorescence lifetimes typically in the region of 0.5 ns to 6 ns, which is much shorter than the 20 ns or greater that is typical for PAHs.…”

“…High bandwidth digital oscilloscopes have also been used to directly measure the fluorescence lifetimes of PAHs but would not be ideal for the detection of proteins due to their shorter decay times. 16 Frequency-domain fluorescence lifetime detection has instead been the preferred technique for PAHs, and could be adapted for proteins, 15 but would require continuous-wave instead of pulsed-excitation sources, and therefore either significantly more sensitive detectors, or much higher excitation powers for signal recovery, and also additional approaches that can account for DC noise sources.…”