Whole‐column fluorescence‐imaged capillary electrophoresis

Abstract

An axially illuminating whole-column fluorescence imaging capillary electrophoresis (CE) experimental setup was developed. A 6 cm long Teflon tube with an inside diameter (ID) of 42 microm was used as separation column. Excitation light of 488 nm from Ar+ laser was introduced to one end of the separation column by an optical fiber. The excitation light propagated inside the separation column by total internal reflection, since the refractive index of the buffer solution was larger than that of the Teflon tube. The fluorescence from the whole separation column was imaged with a charge-coupled device (CCD) camera. Fluorescent compounds such as fluorescein isothiocyanate (FITC), 5-carboxyfluorescein, and FITC-labeled protein were used to test the basic performance of the experimental setup. Experimental results illustrate that the whole-column-fluorescence imaging CE is a fast and sensitive separation method for fluorescent compounds and fluorescent-labeled proteins. Furthermore, it could be used for simple, fast, and easy comparisons of the resistance to photodegradation for various fluorescent compounds.

Recent developments in capillary electrophoresis and capillary electrochromatography of peptides

“…CIEF with the liquid-core waveguide LIF whole-column detection [214], described in more detail in the above Section 6.1 on UV-absorption detection has been applied for separation and analysis of several different proteins and polypeptides, for microheterogeneity characterization and pI determination of antibodies, glycoproteins, and viruses [112,114,115].…”

Study of protein–protein binding reaction by whole‐column fluorescence‐imaged CIEF

“…In order to minimize the inhomogeneous baseline and background along the capillary caused by multi-reflection between the interfaces of the solution and the Teflon tube, the fluorescence images taken after the application of the electric field were divided by that before the application of the electric field [31]. Figure 2 shows the results for GFP.…”

Modified Hadamard transform microchip electrophoresis

“…A whole-column imaging technique uses a detector array to collect the signal that results in an averaged "end" spectrum. In this way, the detection limit is improved by a factor of 3.8 [13][14][15][16] and can be considered as a multiplexing on detector. Only one sample injection is required for col-lecting the multiple signals of the detector, instead of using one detector and multiple injections as in Fourier and Hadamard systems.…”