Such batch measurements of samples with a distribution of R h and a corresponding distribution of diffusion times will yield autocorrelation functions described by the sum of the autocorrelation functions of all particle species in the sample weighed by their light scattering intensities. 3 illustrates how these different measures compare for the compact, globular protein lysozyme.īatch DLS can determine size distributions and the presence of multiple populations in a sample without the need for chromatographic separation, making it an excellent tool for rapid quality control and high-throughput screening of large numbers of samples. Other measures include R g (the radius of gyration, or root-mean-square radius, obtained by e.g., MALS), R vol (the radius of a hypothetical sphere that occupies the same volume as the macromolecule), and R Rot (the radius subtended by rotating the macromolecule). The R h measured by DLS is the radius of a hard sphere with the same diffusion coefficient as the sample. It is therefore important to know how a reported “size” was determined and whether it refers to the radius or diameter of the molecule. Where k is Boltzmann's constant, T is the temperature in K, and h is the solvent viscosity.ĭifferent sizing techniques, e.g., DLS, small angle X-ray scattering, microscopy, and molecular modeling may report different types of radii. The Stokes-Einstein equation then gives the hydrodynamic radius, R h, (Fig. A typical autocorrelation function for a monodisperse sample is shown in Fig. The analysis is done directly in the accompanying DYNAMICS ®, DYNAMICS ® Touch ™ or ASTRA ® software. The analyte’s translational diffusion coefficient, D t, is obtained by automated nonlinear least squares fitting of the autocorrelation function that quantitatively describes the measured time-dependent fluctuations in light scattering intensity. Therefore, the fluctuation in light intensity contains information about the diffusion of the molecules and can be used to extract a diffusion coefficient and calculate a particle size.ĭLS is employed by the DynaPro ® NanoStar ®, the DynaPro ® Plate Reader, the Mobius™ and the WyattQELS ™ module module for MALS detectors to determine the effective particle size. Smaller particles diffuse faster, causing more rapid fluctuations in the intensity than larger particles. The rate of fluctuations is directly related to the rate of diffusion of the molecule through the solvent, which is related in turn to the particles' hydrodynamic radii. In dynamic light scattering (DLS), the time-dependent fluctuations in the scattered light are measured by a single photon counting module. This leads to time-dependent fluctuations in the intensity of the scattered light (Fig. Weeks, Emory University).Īs light scatters from the moving macromolecules, this motion imparts a randomness to the phase of the scattered light, such that when the scattered light from two or more particles is added together, there will be a changing constructive or destructive interference. As can be seen, each particle is constantly moving, and its motion is uncorrelated with the other particles. For example, consider this movie of 2 µm diameter particles in pure water. This leads to a random motion of the molecules called Brownian motion. When in solution, macromolecules are buffeted by the solvent molecules. Dynamic & Electrophoretic Light Scattering.
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