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Related Experiment Video

Updated: Sep 2, 2025

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS
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Method for Improved Fluorescence Corrections for Molar Mass Characterization by Multiangle Light Scattering.

Zachariah A Pittman1, Madeline E McCarthy1, Marc R Birtwistle1

  • 1Clemson University, Chemical and Biomolecular Engineering, Earle Hall, 206 South Palmetto Blvd, Clemson, South Carolina 29631, United States.

Biomacromolecules
|August 4, 2022
PubMed
Summary
This summary is machine-generated.

Accurately measuring the molar mass of fluorescent macromolecules is challenging. This study introduces a new correction method to eliminate fluorescence interference in multiangle light scattering (MALS) measurements, enabling precise characterization.

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Area of Science:

  • Analytical Chemistry
  • Polymer Science
  • Biophysical Chemistry

Background:

  • Multiangle light scattering (MALS) is a standard technique for determining the absolute molar mass of macromolecules.
  • Fluorescent emissions can interfere with MALS measurements, introducing significant errors and hindering accurate characterization of fluorescent macromolecules and particles.
  • Standard bandwidth filters are often insufficient to completely suppress fluorescence in MALS detectors.

Purpose of the Study:

  • To develop and validate a correction procedure to accurately determine the molar mass of fluorescent macromolecules using MALS.
  • To address the challenge of fluorescence interference that standard filtering methods cannot fully eliminate.

Main Methods:

  • Devised a correction procedure to quantify and subtract fluorescence interference from filtered MALS signals.
  • Utilized standard fluorimetry techniques to calculate fluorescence transmission rates before MALS experiments.
  • Synthesized fluorescent dye-conjugated proteins (IR800CW-BSA) to validate the correction procedure.

Main Results:

  • The developed correction procedure successfully eliminated fluorescence interference in MALS measurements.
  • Accurate absolute molar mass determination of fluorescent macromolecules was achieved.
  • Validated the method using fluorescent dye-conjugated proteins.

Conclusions:

  • The proposed correction procedure enables accurate molar mass characterization of fluorescent macromolecules.
  • This method is applicable to various fluorescent materials, including lignins, fluorescent proteins, and nanoparticles.
  • Enhances the reliability of MALS for characterizing samples with intrinsic or introduced fluorescence.