Lamp-based wavelength-resolved fluorescence detection for protein capillary electrophoresis: setup and detector performance.
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.
de Kort
Bregje J
BJ
Department of Biomedical Analysis, Utrecht University, Utrecht, The Netherlands. B.J.deKort@uu.nl
de Jong
Gerhardus J
GJ
Somsen
Govert W
GW
D000818:Animals; D002645:Chickens; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods; D009113:Muramidase / Q000737:chemistry; D055100:Optical Fibers; D010710:Phosphates / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*; D013696:Temperature
D010710:Phosphates; D011506:Proteins; D009113:Muramidase; C018279:sodium phosphate