Six orders of magnitude dynamic range in capillary electrophoresis with ultrasensitive laser-induced fluorescence detection

Abstract

An ultrasensitive laser-induced fluorescence detector was used with capillary electrophoresis for the study of 5-carboxy-tetramethylrhodamine. The raw signal from the detector provided roughly three orders of magnitude dynamic range. The signal saturated at high analyte concentrations due to the dead time associated with the single-photon counting avalanche photodiode employed in the detector. The signal can be corrected for the detector dead time, providing an additional order of magnitude dynamic range. To further increase dynamic range, two fiber-optic beam-splitters were cascaded to generate a primary signal and two attenuated signals, each monitored by a single-photon counting avalanche photodiode. The combined signals from the three photodiodes are reasonably linear from the concentration detection limit of 3 pM to 10 μM, the maximum concentration investigated, a range of 3,000,000. Mass detection limits were 150 yoctomoles injected onto the capillary.

Metabolic Cytometry: Capillary Electrophoresis with Two-Color Fluorescence Detection for the Simultaneous Study of Two Glycosphingolipid Metabolic Pathways in Single Primary Neurons

“…12–14 To address this very wide variation in uptake and metabolic activity, we have created a set of fluorescence detectors with six to nine orders of magnitude dynamic range. 15–16 These instruments have revealed that cultured PC12 cells tend to generate very similar expression profiles of fluorescent glycolipids, which provides confidence in the reproducibility of the analytical method. In constrast, neurons provide a quite diverse population of metabolic products, undoubtedly reflecting differences in their function.…”

Capillary electrophoresis coupled with automated fraction collection

“…Briefly, fluorescence was excited using a CW 532 nm diode-pumped laser (CrystaLaser Model CL532-025), which was focused into the sample stream at the center of a sheath-flow cuvette. Fluorescence was collected at right angles and detected by a cascade of single-photon counting avalanche photodiode modules (PerkinElmer, Montreal, PQ Canada) with a sampling frequency of 50 Hz [23,24]. …”

Capillary isoelectric focusing with pH 9.7 cathode for the analysis of gastric biopsies

“…Our laser-induced fluorescence instrument has been described in detail elsewhere [34–41]. Briefly, a multipurpose injection block was connected to a CZE1000R high-voltage power supply (Spellman).…”

“…The data was processed using SigMan 1.2.6 (http://sourceforge.net/projects/sigman/), and plotted in Matlab (Mathworks). The data were corrected for photodetector nonlinearity before further processing [40–41]. The corrected data were treated with a 5-point median filter to remove spikes caused by the passage of particles through the laser beam and then smoothed by convolution with a Gaussian function that had a 5-point (0.5 s) standard deviation.…”