Characterization of a post-column reaction—laser-induced fluorescence detector for capillary zone electrophoresis

Nickerson, Beverly; Jorgenson, James W.

doi:10.1016/s0021-9673(01)84285-0

Cited by 88 publications

(30 citation statements)

References 23 publications

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“…Multiple products and increased background are additional pitfalls of protein-tagging methodologies [1]. Many of these problems can be resolved by using postcolumn derivatization [8]. However, the improvement in sensitivity gained by using postcolumn techniques is offset by instrumental complexity as well as the dependence of peak efficiency upon reagent flow rate and reaction distance [9].…”

Section: Introductionmentioning

confidence: 99%

Light-emitting diode-induced fluorescence detection of native proteins in capillary electrophoresis

Sluszny

Yeung

2005

Electrophoresis

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A continuous-wave 280 nm light-emitting diode (LED) was used as the excitation source for native fluorescence detection of proteins in CE. The operating current and temperature of the LED were optimized in order to achieve high luminescence power. It was found that a forward current of 30 mA and a temperature of approximately 5 degrees C gave the best S/N. By using a set of two ball lenses to focus light from the LED, we achieved a spot of approximately 200 mum with a power of 0.1-0.2 mW on the detection window. Fluorescence was collected with a ball lens at 90 degrees angle through a bandpass filter onto a photomultiplier tube. In CZE an LOD of 20 nM for conalbumin was reached. In capillary gel electrophoresis all eight proteins from a commercial standard kit were detected with high S/N. For a 10 microg/mL total protein mixture, S/N was better than 3 for all proteins in solution. Further improvement in LOD should be possible on utilization of an LED with higher luminescence power.

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Section: Introductionmentioning

confidence: 99%

Light-emitting diode-induced fluorescence detection of native proteins in capillary electrophoresis

Sluszny

Yeung

2005

Electrophoresis

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“…Since this cell can be the most significant contributor to extra-column broadening [15], alternate detection modes have also been surveyed. On-column detection has been employed to eliminate extra tubing requirements, but the low path length tends to hurt sensitivity [56,[79][80][81][82][83] and similar measurements using fluorescence require derivatization for many analytes [84][85][86][87]. An alternative option with a low limit of detection and reduced volume is electrochemical detection with a microfiber electrode [49,[88][89][90].…”

Section: Detector Contributionsmentioning

confidence: 99%

Measurement and Modeling of Extra-Column Effects Due to Injection and Connections in Capillary Liquid Chromatography

et al. 2015

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Abstract:As column volumes continue to decrease, extra-column band broadening has become an increasingly important consideration when determining column performance. Combined contributions due to the injector and connecting tubing in a capillary LC system were measured and found to be larger than expected by Taylor-Aris theory. Variance from sigma-type and tau-type broadening was isolated from eluted peaks using the Foley-Dorsey Exponentially Modified Gaussian peak fitting model and confirmed with computational fluid dynamics. It was found that the tau-type contributions were the main cause for the excessive broadening because of poorly-swept volumes at the connection between the injector and tubing. To reduce tau-type contributions (and peak tailing), a timed pinch mode could be used for analyte injection.

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“…This underscores the sensitivity of laser fluorimetric detection, but has a limited interest for actual applications in biological fluids having low analyte concentrations. At low concentrations, slow reaction rate and strong analyte adsorption may occur yielding incomplete reaction with formation of a vast number of fluorescent side-products having different charge-to-size ratios [84] and giving multiple or broad peaks and noisy baselines.…”

Section: Detection Of Nonfluorescent Peptides and Proteinsmentioning

confidence: 99%

“…The formed derivatives may be subjected to some degradation which explains why OPA is mainly used in postcolumn derivatization. In this case, it gives LODs at attomole levels for proteins [84]. Using OPA labelling, it was possible to analyse the content of Hb and carbonic anhydrase I in a single cell of human erythrocytes isolated from whole blood samples [90] with a coaxial postcolumn reactor fitted with a 30 mm id reaction capillary, itself connected to a 15 mm id capillary for separation.…”

Section: Ortho-phthalaldehyde (Opa)mentioning

confidence: 99%

LIF detection of peptides and proteins in CE

2007

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CE- and microchip-based separations coupled with LIF are powerful tools for the separation, detection and determination of biomolecules. CE with certain configurations has the potential to detect a small number of molecules or even a single molecule, thanks to the high spatial coherence of the laser source which permits the excitation of very small sample volumes with high efficiency. This review article discusses the use of LIF detection for the analysis of peptides and proteins in CE. The most common laser sources, basic instrumentation, derivatization modes and set-ups are briefly presented and special attention is paid to the different fluorogenic agents used for pre-, on- and postcapillary derivatization of the functional groups of these compounds. A table summarizing major applications of these derivatization reactions to the analysis of peptides and proteins in CE-LIF and a bibliography with 184 references are provided which covers papers published to the end of 2005.

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Characterization of a post-column reaction—laser-induced fluorescence detector for capillary zone electrophoresis

Cited by 88 publications

References 23 publications

Light-emitting diode-induced fluorescence detection of native proteins in capillary electrophoresis

Light-emitting diode-induced fluorescence detection of native proteins in capillary electrophoresis

Measurement and Modeling of Extra-Column Effects Due to Injection and Connections in Capillary Liquid Chromatography

LIF detection of peptides and proteins in CE

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