Lamp‐based wavelength‐resolved fluorescence detection for protein capillary electrophoresis: Setup and detector performance

Abstract

A lamp-based fluorescence detection (Flu) system for CE was extended with a wavelength-resolved (WR) detector to allow recording of full protein emission spectra. WRFlu was achieved using a fluorescence cell that employs optical fibres to lead excitation light from a Xe-Hg lamp to the capillary window and protein fluorescence emission to a spectrograph equipped with a CCD. A 280 nm band pass filter etc. together with a 300 nm short pass cut-off filter was used for excitation. A capillary cartridge was modified to hold the detection cell in a commercial CE instrument enabling WRFlu in routine CE. The performance of the WRFlu detection was evaluated and optimised using lysozyme as model protein. Based on reference spectral data, a signal-intensity adjustment was introduced to correct for transmission losses in the detector optics that occurred for lower protein emission wavelengths. CE-WRFlu of lysozyme was performed using BGEs of 50 mM sodium phosphate (pH 6.5 or 3.0) and a charged-polymer coated capillary. Using the 3-D data set, signal averaging over time and emission-wavelength intervals was carried out to improve the S/N of emission spectra and electropherograms. The detection limit for lysozyme was 21 nM, providing sufficient sensitivity to obtain spectral information on protein impurities.

Capillary Electrophoresis with Lamp-Based Wavelength-Resolved Fluorescence Detection for the Probing of Protein Conformational Changes

“…However, for reliable determination of the maximum emission wavelength, the RSD should be below 0.3% (i.e., 1.0 nm), which is within the spectral resolution of the detector. 19 So, taking the data in Tables 1 and 2 into account, we conclude that for carbonic anhydrase II, β-lactoglobulin B, and lysozyme concentrations should be about 15 times and for R-chymotrypsinogen A about 28 times above their LOD to allow adequate determination of their maximum emission wavelength with the CE-wrFlu system.…”

Chiral capillary electrophoresis with UV-excited fluorescence detection for the enantioselective analysis of 9-fluorenylmethoxycarbonyl-derivatized amino acids

“…In order to achieve appropriate UV excitation, a CE-dedicated fluorescence detector equipped with a Xe-Hg source was employed [55]. Previously, this system has shown useful for measuring native protein emission upon UV excitation [57]. The system comprises a spectrograph and CCD allowing on-line wavelength-resolved fluorescence (wrFlu) detection.…”

“…A previously described wavelength-resolved fluorescence (wrFlu) detector for CE was used, which was based on an Argos 250B fluorescence detection cell (Flux Instruments, Basel, Switzerland) [55] combined with a SR-163 spectrograph equipped with a CCD camera (Andor Technologies, Darmstadt, Germany) [56, 57]. The Argos system comprises a Xe-Hg lamp for excitation, excitation, and emission optical guides and filters and an optical cone detection cell.…”

Enantioselective micellar electrokinetic chromatography of dl‐amino acids using (+)‐1‐(9‐fluorenyl)‐ethyl chloroformate derivatization and UV‐induced fluorescence detection

“…As described by de Kort et al. , this difference is caused by a somewhat reduced transmittance of UV wavelengths below 315 nm of the detector optics (optical cone and emission fiber).…”