Impact of laser excitation intensity on deep UV fluorescence detection in microchip electrophoresis.
A high intensity 266 nm continuous wave (cw-) laser developed for material processing was utilised as an excitation source for sensitive native fluorescence detection of unlabelled compounds in MCE. This 120 mW laser was attached via an optical fibre into a commercial epifluorescence microscope. With this MCE set-up we evaluated the impact of laser power on the S/N of aromatic compounds as well as of proteins. Compared with a previous work which used a 4 mW pulsed laser for excitation, improved S/N for small aromatics and to a lesser extent for proteins could be attained. The LOD of the system was determined down to 24 ng/mL for serotonin (113 nM), 24 ng/mL for propranolol (81 nM), 80 ng/mL for tryptophan (392 nM) and 80 ng/mL for an aromatic diol (475 nM). Sensitive protein detection was obtained at concentrations of 5 microg/mL for lysocyme, trypsinogen and chymotrypsinogen (340, 208 and 195 nM, respectively). Finally, a comparison of the cw- with a pulsed 266 nm laser, operating at the same average power, showed a higher attainable sensitivity of the cw-laser. This can be attributed to fluorescence saturation and photobleaching effects of the pulsed laser at high pulse energies.
Schulze
Philipp
P
Institute of Analytical Chemistry, University of Leipzig, Leipzig, Germany.
Ludwig
Martin
M
Belder
Detlev
D
D046209:Electrophoresis, Microchip / Q000295:instrumentation* / Q000379:methods; D007834:Lasers; D011506:Proteins / Q000032:analysis*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods; D014466:Ultraviolet Rays
D011506:Proteins