In dynamic light scattering, it is the Brownian motion of the particles that is measured, not the size. The size is calculated from the measured diffusion coefficient, using the Stokes-Einstein equation and the known viscosity of the medium. As such, any factor which influences the motion of the particles, and hence the measured diffusion coefficient, will also influence the subsequently calculated hydrodynamic size.
Examples of Brownian motion influencing factors are temperature, medium viscosity, and sample concentration. Temperature and viscosity are easily measured and accounted for within the Stokes-Einstein equation. Sample concentration effects however, are much more complex, and at higher sample concentrations, multiple measurements at different concentrations are typically required to identify and correct for sample concentration effects. Alternately, sample concentration effects can be minimized by conducting DLS measurements in the limit of infinite dilution – with the conundrum being, that as the sample is diluted, the signal to noise ratio and hence the confidence in the resultant size is diminished. So as you minimize sample concentration effects, you also reduce confidence in the results.
ISO 13321, entitled "Particle Size Analysis – Photon Correlation Spectroscopy", recommends using a Dynamic Debye Plot for measuring particle size by DLS in the absence of sample concentration effects. The procedures for generating a Dynamic Debye plot are:
1) measure the diffusion coefficient (D) at several concentrations
2) plot the diffusion coefficient dependence on concentration
3) extrapolate to zero concentration to determine the diffusion coefficient (Do) in the limit of infinite dilution.
The particle size calculated from the diffusion coefficient (Do) measured in the limit of infinite dilution is defined as the hydrodynamic size or Z average if determined using a Cumulants analysis. Figure 1 shows an example of a Dynamic Debye plot for lysozyme in 500 and 10 mM NaCl. The diffusion coefficients in the limit of infinite dilution are 125 um2/s (500 mM NaCl) and 126 um2/s (10 mM NaCl), both of which yield a hydrodynamic diameter of 3.7 nm for lysozyme.
Figure 1: Dynamic Debye plot for lysozyme in 500 and 10 mM NaCl.
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