2.5.  Static and dynamic light scattering (SLS/DLS)

Light scattering refers to a physical process involving the interaction of light with a small particle or molecule. When an incident photon encounters a molecule, it perturbs the electron orbits within the molecule and creates an induced dipole moment, which becomes a source of electromagnetic radiation with energy radiated in all directions. The evaluation of the scattered light with regard to its intensity and wavelength often yields valuable information about the scattering molecules, such as molar mass, binding affinity, and absolute stoichiometry of complex interactions. This makes light scattering a versatile tool for characterization of macromolecules and their interactions in solution.58 In contrast to most methods for characterization, light scattering is an absolute measurement without requiring outside calibration standards.

The most widely used light scattering techniques are static light scattering (SLS) and dynamic light scattering (DLS). In SLS, also known as classical light scattering or multiple angle light scattering, time‐averaged scattered light intensity is collected at multiple different angles, with weight average molar mass, size and shape of molecules, and second virial coefficient reflecting PPIs calculated from this data. In DLS, also known as quasi‐elastic light scattering, the fluctuations in the intensity of scattered light at a certain angle as the effect of the Brownian motion of the scattering molecules is detected, yielding information about translational diffusion coefficient and hydrodynamic radius of molecules.59 Combining a light scattering detection technique and a separation system, such as size‐exclusion chromatography, provides a good method for the semi‐quantitative characterization of PPIs with high sensitivity and high resolution in short run times.58, 60 As interaction studies should be performed under true equilibrium, a series of batch SLS or DLS measurements over varying compositions referred to as composition‐gradient SLS (CG‐SLS) or DLS (CG‐DLS) have been used to determine equilibrium properties including association stoichiometry and binding affinity of interacting macromolecules in solution.61, 62