Continuous wave-based multiphoton excitation fluorescence for capillary electrophoresis

Area of Science:

• Analytical Chemistry
• Biophysical Chemistry
• Separation Science

Background:

• Capillary electrophoresis (CE) is a powerful separation technique.
• Sensitive detection methods are crucial for analyzing small sample volumes in CE.
• Conventional laser-induced fluorescence (LIF) detection has limitations in sensitivity and multiplexing.

Purpose of the Study:

• To introduce and evaluate a novel continuous wave (CW)-based multiphoton excitation (MPE) fluorescence detection method with a diode laser (DL) for CE.
• To compare the performance of end-column versus on-column detection configurations.
• To assess the applicability of this method for various fluorescent labels and separation modes.

Main Methods:

• Development of a CW-MPE fluorescence detection system with a DL for CE.
• Utilized an end-column detection configuration for improved sensitivity.
• Evaluated small-molecule dyes, fluorescent proteins, and quantum dots (QDs) as fluorescent tags.
• Performed quantitative analysis using rhodamine species and separation of FITC-labeled amino acids using CZE and MEKC.

Main Results:

• The end-column detection configuration demonstrated superior sensitivity compared to on-column detection.
• Achieved a dynamic linear range over two orders of magnitude with detection limits down to the zeptomole level for rhodamine species.
• Demonstrated simultaneous detection of multiple fluorescent dyes with different spectral properties.
• Successful separation and detection of FITC-labeled amino acids in CZE and MEKC.

Conclusions:

• The proposed CW-MPE fluorescence detection with DL is a highly sensitive and versatile method for CE.
• This technique offers advantages over conventional LIF detection, particularly for simultaneous multi-analyte detection.
• The method shows significant potential for applications in micro/nano-column liquid phase separation technologies and bioanalysis.