14 Buyers’ Guide 2021
Expanding the Boundaries of Light Scattering for Macromolecules
Sébastien Rouzeau, Tosoh Bioscience GmbH, Griesheim, Germany
sebastien.rouzeau@
tosoh.com
In the past four decades, light scattering detection (LS) has become a widely used technique to obtain the supposed “absolute” or true molecular weight of macromolecules such as synthetic polymers, biopolymers, proteins, and antibodies. When coupled with size exclusion chromatography (SEC), molecular weight and size, as well as aggregation or branching can be investigated to provide analytical scientists with a powerful characterisation tool for these large molecules.
Whether new biopharmaceutical products or polymers with tailored properties, the macromolecules’ complexity increases, requiring even more extensive and in-depth characterisation for a full understanding of the material. With limited amounts of costly samples available for analysis, analytical scientists are facing the challenge of fi nding new technologies that help them improve and optimise their analytical characterisation.
The latest light scattering technology introduced by Tosoh Bioscience features an entirely new detector design that addresses the limitations and shortcomings of the current, state-of-the-art LS instruments.
Principles and theory of light scattering
When a beam of light illuminates molecules in a solvent, oscillating dipoles are generated within the molecules, re-emitting a small amount of light in all directions. This physical phenomenon shows two main properties. Firstly, the intensity of the scattered light is directly related to the molecular weight of the molecules: higher molecular weight molecules scatter more light. Secondly, the scattered light’s intensity is not identical in all directions, and this dissymmetric scattering pattern is related to the size and shape of the molecules.
Consequently, molecular weight information is obtained from the intensity of the scattered light, while molecular size is obtained by examining intensity changes with the angle of observation.
Rayleigh defi ned the theory and equations of light scattering by sub-micron particles. He established the relationship of the intensity of scattered light to the molecular weight and size of the molecules, the concentration of the solution, and the measurement angle [1].
Rayleigh determined the molecular weight of a molecule can be obtained directly from the intensity of the scattered light in the same direction as the incident beam (0° angle), regardless of the molecule’s size and shape.
However, due to the incident beam, measuring the amount of scattered light at 0° angle is technically impossible. Light scattering instruments nevertheless have to work around this technical challenge to provide the molecular weight information.
Figure 1: LenS3
Traditional light scattering instruments and their limitations
The fi rst LS instruments, in the 1970’s, used a low angle light scattering approach (LALS) in which the angle of measurement is close enough to 0° to assume safely that the measurement at such a low angle is the same as the theoretical 0° measurement. This is still considered the most accurate molecular weight measurement method, as it requires no additional assumptions or extrapolations. However, those early LALS instruments were not easy to use and a low angle measurement alone does not provide size information since it does not measure the angular dependence of the scattered
fl ow channel. light intensity.
To achieve both easier molecular weight and size measurements simultaneously, multi-angle light scattering (MALS) detectors were developed to collect light at multiple angles. These measurements are extrapolated back to 0° for molecular weight (MW) determination, while the scattering pattern is mapped to obtain size information described as the radius of gyration (Rg
).
MALS instruments typically consist of multiple photodiodes (detectors) arranged in the same plane, around a circular or cylindrical fl ow cell. There are several technical limitations to this design since the geometry of this design offers limited space available for the physical location
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