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Related Experiment Videos

Quantitative measurements on wetted thin layer chromatography plates using a charge coupled device camera.

Michael Lancaster1, David M Goodall, Edmund T Bergström

  • 1Department of Chemistry, University of York, York YOI0 5DD, UK.

Journal of Chromatography. A
|October 1, 2005
PubMed
Summary

This study introduces imaging wet spots on thin-layer chromatography (TLC) plates, enabling accurate quantification of Sudan II. Wet imaging offers a linear relationship between peak area and sample loading, comparable to dry methods.

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Area of Science:

  • Analytical Chemistry
  • Chromatography
  • Spectroscopy

Background:

  • Thin-layer chromatography (TLC) is a widely used separation technique.
  • Quantification of analytes on TLC plates typically requires drying before imaging.
  • Imaging wet spots could potentially streamline the TLC analysis process.

Purpose of the Study:

  • To investigate the feasibility and accuracy of imaging and quantifying analytes on thin-layer chromatographic plates while they are still wet with solvent.
  • To compare the quantitative performance of wet-plate imaging with traditional dry-plate imaging.

Main Methods:

  • Utilized a charge-coupled device (CCD) camera for imaging Sudan II spots on TLC plates.
  • Performed imaging in both reflectance and transmission modes after development with dichloromethane.

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  • Applied the Beer-Lambert equation for data processing, incorporating an empirical expression for high loadings to account for scattering effects.

    • Main Results:

    • Established a linear relationship (r² > 0.99) between peak area and sample loading for both wet and dry modes over an order of magnitude.
    • Observed that the limit of detection was lower for dry plates (0.5 ng) compared to wet plates (2 ng).
    • Achieved good precision, with 1-4% RSD intra-plate and 8-14% RSD inter-plate.

    Conclusions:

    • Imaging wet TLC plates is a viable method for quantification, demonstrating linearity comparable to dry plates.
    • While dry plate imaging offers a lower limit of detection, wet imaging provides a potentially faster analytical workflow.
    • The study validates the use of Beer-Lambert and Kubelka-Munk treatments for analyzing wet TLC plates, considering scattering effects.