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Fluorescence Enhancement of Pyrene Measured by Thin-Layer Chromatography with Diode-Array Detection

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Summary

In-situ densitometry for qualitative or quantitative purposes is a key step in thin-layer chromatography. It offers a simple way of quantifying by measuring the optical density of the separated spots directly on the plate. A new TLC scanner has been developed which is able to measure TLC plates or HPTLC plates, at different wavelengths simultaneously, without destroying the plate surface. The system enables absorbance and fluorescence measurements in one run. Fluorescence measurements are possible without filters or other adjustments.

The measurement of fluorescence from a TLC plate is a versatile means of making TLC analysis more sensitive. Fluorescence measurements with the new scanner are possible without filters or special lamps. Improvement of the signal-to-noise ratio is achieved by wavelength bundling. During plate scanning the scattered light and the fluorescence are both emitted from the surface of the TLC plate and this emitted light provides the desired spectral information from substances on the TLC plate. The measurement of fluorescence spectra and absorbance spectra directly from a TLC plate is based on differential measurement of light emerging from sample-free and sample-containing zones.

The literature recommends dipping TLC plates in viscous liquids to enhance fluorescence. Measurement of the fluorescence and absorbance spectra of pyrene spots reveals the mechanism of enhancement of plate dipping in viscous liquids—blocked contact of the fluorescent molecules with the stationary phase or other sample molecules is responsible for the enhanced fluorescence at lower concentrations.

In conclusion, dipping in TLC analysis is no miracle. It is based on similar mechanisms observable in liquids. The measured TLC spectra are also very similar to liquid spectra and this makes TLC spec-troscopy an important tool in separation analysis.

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Authors and Affiliations

  1. University of Applied Sciences Offenburg, Badstrasse 24, D-77652, Offenburg, Germany

    Bernd Spangenberg & Klemens Lorenz

  2. Lehrstuhl für Molekulare Physikalische Chemie, Universität Karlsruhe, Kaiserstrasse 12, D-76128, Karlsruhe, Germany

    Steffen Nasterlack

Authors
  1. Bernd Spangenberg
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  2. Klemens Lorenz
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  3. Steffen Nasterlack
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Spangenberg, B., Lorenz, K. & Nasterlack, S. Fluorescence Enhancement of Pyrene Measured by Thin-Layer Chromatography with Diode-Array Detection. JPC-J Planar Chromat 16, 331–337 (2003). https://doi.org/10.1556/JPC.16.2003.5.1

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  • DOI: https://doi.org/10.1556/JPC.16.2003.5.1

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